Compositions and methods for increasing telomerase activity

ABSTRACT

The present invention relates to methods and compositions for increasing telomerase activity in cells. Such compositions include pharmaceutical formulations. The methods and compositions are useful for treating diseases subject to treatment by an increase in telomerase activity in cells or tissue of a patient. They are also useful for enhancing replicative capacity of cells in culture, as in ex vivo cell therapy and for enhancing proliferation of stem and progenitor cells.

REFERENCE TO PRIOR APPLICATIONS

This application is a continuation of and claims the benefit to U.S.application Ser. No. 12/781,515 filed May 17, 2010, which claims thebenefit of U.S. Provisional Application No. 61/179,305 filed May 18,2009, each of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for increasingtelomerase activity in cells.

BACKGROUND OF THE INVENTION

Telomerase is a ribonucleoprotein that catalyzes the addition oftelomeric repeats to the ends of telomeres. Telomeres are long stretchesof repeated sequences that cap the ends of chromosomes and are believedto stabilize the chromosome. In humans, telomeres are typically 7-10 kbin length and comprise multiple repeats of the sequence -TTAGGG-.Telomerase is not expressed in most adult cells, and telomere lengthdecreases with successive rounds of replication. After a certain numberof rounds of replication, the progressive shortening of the telomeresresults in the cells entering a telomeric crisis stage, which in turnleads to cellular senescence. Certain diseases are associated with rapidtelomeric loss, resulting in premature cell senescence. Expression ofthe gene encoding the human telomerase protein in human cells has beenshown to confer an immortal phenotype, presumably through bypassing thecells' natural senescence pathway. In addition, expression of thetelomerase gene in aging cells with short telomeres has been shown toproduce an increase in telomere length and restore a phenotype typicallyassociated with younger cells.

Somatic cells, in contrast to tumor cells and certain stem cells, havelittle or no telomerase activity and stop dividing when the telomericends of at least some chromosomes have been shortened to a criticallength, leading to programmed cellular senescence (cell death). Sincethe loss of telomeric repeats in somatic cells, leading to senescence,is augmented by low telomerase activity, induction of telomeraseactivity, which has the effect of adding arrays of telomeric repeats totelomeres, thereby imparts to mortal somatic cells increased replicativecapacity, and imparts to senescent cells the ability to proliferate andappropriately exit the cell cycle upon repair of damaged tissue.

Methods of increasing telomerase activity therapeutically have beeninvestigated by, for example, Bodnar Science 279(5349):349-52 (Jan. 16,1998)); White, PCT Int. Appl. Pubn. No. WO 2000/08135 (February 2000));Hannon et al. PCT Int. appl. Pubn. WO 99/35243 (July 1999) and PCT Int.Appl. Pubn. No. WO 2000/0312238 (June 2000)); Franzese et al.Lifescience 69(13) 1509-20 (2001), and Yudoh et al. J. Bone and MineralRes. 16(8):1453-1464 (2001). In these reports, telomerase activity isgenerally increased by overexpression of hTERT, the gene encoding theprotein component of human telomerase, or by expression of proteinswhich mediate assembly of telomerase, e.g. heat shock proteins (White,PCT No. WO2000/08135). Franzese et al. reported that Saquinavir, aprotease inhibitor prescribed for treatment of HIV infection, increasedtelomerase activity in peripheral blood mononuclear cells; Vasa et al.Circ Res, 87(7) 540-2 (2000) described activation of telomerase, and aresulting delay in endothelial senescence, by administration of a nitricoxide (NO) precursor.

Various saponins of the astragaloside family have been reported ashaving various biological effects including increasing telomeraseactivity, Harley et al. PCT Int Appl. Pubn. No. WO2005/000245. It wouldbe beneficial to develop a compound which was an effective telomeraseactivator.

SUMMARY OF THE INVENTION

The invention described herein is generally related to compounds andmethods for increasing telomerase activity in cells and compositions foruse in such methods. Such methods and compositions may be used on cellsin cell culture, i.e. in vitro or ex vivo, or in vivo, such as cellsgrowing in tissues of a subject, including human subjects and non-humanmammals.

Various saponins of the astragaloside family had previously beenreported as having various biological effects including increasingtelomerase activity, Harley et al. PCT Int Appl. Pubn. No.WO2005/000245. However, the inventors have found that thebioavailability of the naturally occurring compounds described thereinincluding cycloastragenol is very limited when administered orally tocertain mammalian species. It was not clear whether the limitedbioavailability was attributable to low uptake of the compounds by themammals, or high metabolism of the compounds in certain species ofmammals or a combination of both. Such low bioavailability means thatthe compounds previously described were very much less effective as anoral telomerase activator in certain mammalian species.

It was determined that there was a need for a new compounds which wouldbe potent telomerase activators and which were also orally availableacross a number of mammalian species and which had an improved half lifein representative mammalian species. The chemical compounds describedherein possess these desired properties.

In particular embodiments, the compositions comprise a compound offormula I and pharmaceutical salts thereof as described below. Aspectsof the invention include formulations of such compounds for use inpharmaceutical applications, in particular in applications whereincreasing telomerase activity in cells is shown to be, or expected tobe, beneficial. Methods of using the compounds and formulations thereoffor such applications are also provided, including methods for applyingor administering such formulations after the need for, or advantage of,increasing telomerase activity in cells or tissues has been determined.

The present invention includes, in one aspect, a compound the formula I:

wherein X¹, is selected from keto (═O), hydroxy (—OH), and

wherein X² is selected from keto (═O), hydroxy (—OH), and

wherein X³ is selected from keto (═O), hydroxy (—OH), and

wherein at least one of X¹, X² or X³ is

respectively;

-   wherein R¹ or R² are independently selected from —CH(CH₃)₂, and    —CH(CH₃)CH₂CH₃;    -   and pharmaceutically acceptable salts thereof.

In one embodiment X¹ is —OC(O)CH(NH2)R¹ wherein R¹ is selected from thegroup consisting of —CH(CH₃)₂ or —CH(CH₃)₂CH₃ In another embodiment X²is —OC(O)CH(NH₂)R² wherein R² is selected from the group consisting of—CH(CH₃)₂ or —CH(CH₃)₂CH₃

In one embodiment at least one of X¹, X² or X³ is —OC(O)CH(NH₂)CH(CH₃)₂.In another embodiment both X¹ and X² are —OC(O)CH(NH₂)CH(CH₃)₂.

In one embodiment at least one of X¹ or X² is—OC(O)CH(NH₂)CH(CH₃)CH₂CH₃. In another embodiment both X¹ and X² are—OC(O)CH(NH₂)CH(CH₃)CH₂CH₃.

In selected embodiments of formula I, X¹ is a —OC(O)CH(NH₂)CH(CH₃)₂ andX² and X³ are independently selected from hydroxy and keto. In furtherembodiments, X² is —OC(O)CH(NH₂)CH(CH₃)₂ and X¹ and X³ are independentlyselected from hydroxy and keto. In further embodiments, X³ is—OC(O)CH(NH₂)CH(CH₃)₂ and X¹ and X² are independently selected fromhydroxy and keto. In further embodiments, X¹ and X² are both—OC(O)CH(NH₂)CH(CH₃)₂ and X³ is OH. In still further embodiments, X¹ is—OC(O)CH(NH₂)CH(CH₃)₂ and each of X² and X³ are OH. In still furtherembodiments, X² is —OC(O)CH(NH₂)CH(CH₃)₂ and each of X¹ and X³ are OH.

In selected embodiments of formula I, X¹ is a —OC(O)CH(NH₂)CH(CH₃)CH₂CH₃and X² and X³ are independently selected from hydroxy and keto. Infurther embodiments, X² is —OC(O)CH(NH₂)CH(CH₃)CH₂CH₃ and X¹ and X³ areindependently selected from hydroxyl and keto. In further embodiments,X¹ and X² are both —OC(O)CH(NH₂)CH(CH₃)CH₂CH₃ and X³ is OH. In stillfurther embodiments, X¹ is —OC(O)CH(NH₂)CH(CH₃)CH₂CH₃ and each of X² andX³ are OH. In still further embodiments, X² is—OC(O)CH(NH₂)CH(CH₃)CH₂CH₃ and each of X¹ and X³ are OH.

In some embodiments of formula I, the pharmaceutically acceptable saltis a hydrochloride salt.

It is contemplated that the amino acid substituents are the L ornaturally occurring stereoisomer.

Exemplary compounds of formula I include those designated herein as:2-(L)-amino-3-methyl-butyric acid6α,16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester (designated herein as 4); 2-(L)-amino-3-methyl-butyric acid6α(2-amino-3-methyl-butyryloxy)-16β-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester (designated herein as 7);2-(L)-tert-butoxycarbonylamino-3-methyl-butyric acid3b-acetoxy-16b-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-6a-ylester (designated herein as 12) 2-(L),3-dimethyl-pentanoic acid6α,16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester (designated herein as 14); 2-(L)-Amino-3-methyl-butyric acid,16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-3-oxo-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-6α-ylester (designated herein as 30), 2-(L)-Amino-3-methyl-pentanoic acid6α-(2-amino-3-methyl-pentanoyloxy)-16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester (designated herein as 32); 2-(L)-Amino-3-methyl-butyric acid3β,6α-dihydroxy-16β-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-16β-ylester (designated herein as 36) and pharmaceutically acceptable saltsthereof.

Exemplary compounds of formulas I include those designated herein as:2-(L)-amino-3-methyl-butyric acid6α,16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester hydrochloride salt; 2-(L)-amino-3-methyl-butyric acid6α-(2-amino-3-methyl-butyryloxy)-16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester hydrochloride salt;2-(L)-tert-butoxycarbonylamino-3-methyl-butyric acid3b-acetoxy-16b-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-6a-ylester hydrochloride salt; 2-(L),3-dimethyl-pentanoic acid6α,16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester hydrochloride salt, 2-(L)-Amino-3-methyl-butyric acid,16β-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-3-oxo-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-6α-ylester hydrochloride salt; 2-(L)-Amino-3-methyl-pentanoic acid,6α-(2-amino-3-methyl-pentanoyloxy)-16β-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester hydrochloride salt or 2-(L)-Amino-3-methyl-butyric acid, 3β,6α-dihydroxy-16β-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-16β-ylester hydrochloride salt.

A compound of formula I above, when formulated in a solvent, iseffective to produce a level of telomerase activity in keratinocytes orPBMCs, as measured in a TRAP assay, at least 50% greater, at least 70%greater, at least 80% greater, or at least 90% greater than the level insaid cells treated with said solvent, as measured in a TRAP assay asdescribed herein. In further embodiments, the compound is effective toproduce a level of telomerase activity in keratinocytes or PBMCs, asmeasured in a TRAP assay, at least 100% greater than the level in saidcells treated with said solvent, as measured in a TRAP assay asdescribed herein.

The present invention includes, in one aspect, a method of increasingtelomerase activity in a cell or tissue. The method comprises contactingthe cell or tissue with an isolated compound of formula I. The methodmay further comprise the preliminary step of identifying a cell ortissue in which an increase in telomerase activity is desired.

The method of contacting an isolated compound of formula I with a cellor tissue may comprise, prior to said contacting, identifying a cell ortissue in which an increase in telomerase activity is desired. Benefitsto be realized by increasing telomerase activity in a cell or tissueinclude, for example, enhancement of the replicative capacity and/orlife span of said cell or cells within said tissue.

The method may include identifying, determining or diagnosing acondition in a subject such that increasing telomerase activity in thecells or tissue of the subjects is desired, and administering thecompound to the subject. The subject is a mammalian subject, such as adomestic animal such as a dog, cat, mouse, rat, monkey or a humansubject or patient.

Such conditions or diseases for prevention or treatment may include, forexample, viral and opportunistic infections including HIV, variousdegenerative diseases, such as neurodegenerative disease, degenerativedisease of the bones or joints, and connective tissues, maculardegeneration, diabetic retinopathy, cardiovascular diseases includingcentral and peripheral vascular disease, Crohn's disease and otherimmunological conditions, liver diseases including fibrosis andcirrhosis, lung diseases including pulmonary fibrosis, asthma,emphysema, and COPD, hematopoietic disorders (including anemia,thrombocytopenia, neutropenia and other cytopenias), chronicinflammatory gastrointestinal diseases such as Barretts esophagus, anydisorder related to loss of proliferative capacity in stem cell orprogenitor cell populations. Such conditions may include bone marrowfailure syndrome, aplastic anemia, myelodysplastic anemia ormyelodysplastic syndrome. Such conditions also include wounds or otheracute or chronic conditions of the skin and its appendages, such as, forexample, a burn, an abrasion, an incision, a graft, a lesion caused byan infectious agent, a chronic venous ulcer, a diabetic ulcer, acompression or decubitus ulcer, a mucosal ulcer, keloid formation, hairor pigment loss, and other structural aberrations of the skin and itsappendages. Such conditions also include cancer and precancerousconditions in which low telomerase or shortened telomeres are associatedwith genomic instability, or increased mutation rates, or loss of tumorsuppressor functions, and consequently subjects have an increased riskof tumor initiation, tumor progression, or tumor recurrence.

The invention provides methods of preventing or treating a condition ina patient, such as those noted above, by increasing telomerase activityin cells or tissue of the patient, the method comprising administeringto a patient in need of such prevention or treatment, an isolatedcompound of formula I as defined above. The compositions may beadministered by various routes, for example, orally, topically,parenterally, subcutaneously, inhalation and intravenously.

In a further embodiment, the invention provides a method of treating anacute or chronic condition of the epidermis, comprising contactingepidermal cells with a topical formulation of an isolated compound offormula I as defined above.

The cells with which the formulation is contacted may also includeexplant cells which are contacted ex vivo, e.g. for cell-basedtherapies, or other cells in culture. Accordingly, the inventionprovides a method of enhancing replicative capacity and improvedfunctional capacity of cells in vitro or ex vivo, comprising contactingsaid cells with an effective amount of a composition comprising acompound of formula I as defined above, including selected embodimentsof the compounds as defined above. In general, the cells are mammaliancells; in selected embodiments, the cells are stem cells, such as bonemarrow stem or progenitor cells, bone marrow stromal cells, epidermaland epithelial stem cells from skin and other tissues including gut,liver, and pancreas, islet precursor cells, neurosphere cells,adrenocortical cells, muscle satellite cells, mesenchymal stem andprogenitor cells including osteoblast precursors, retinal pigmentedepithelial cells, endothelial precursor cells, pericytes, and immunecells capable of clonal expansion including memory and naïve T (CD4 andCD8) and B cells.

In a further embodiment, the invention provides a method of enhancingtransplantation of a tissue from a living donor or cadaver to a livingpatient comprising contacting the transplantation tissue with anisolated compound of formula I as defined above. In a furtherembodiment, the invention provides a method of enhancing transplantationof a tissue from a donor to a living patient comprising administeringthe isolated compound of formula I as defined above to the patienteither before, simultaneous with, or for a period of time after thetransplantation of the tissue. The transplanted tissue may be solidtissue, such as a kidney, heart, lungs etc., or hematopoietic tissuesuch as, without limitation, blood cells such as leukocytes, lymphocytesor hematopoietic precursor cells which may be derived fro bone marrow.

In one embodiment, the invention provides a pharmaceutical compositioncomprising, in a pharmaceutically acceptable vehicle, a compound offormula I as depicted above.

In another embodiment, the invention provides a topical pharmaceuticalformulation of an isolated compound of formula I, as defined above.Selected embodiments of an isolated compounds are also defined above.The topical formulation typically comprises one or more componentsselected from the group consisting of an emulsifier, a carrier (e.g.liposomes), a thickener, and a skin emollient. Such compositions may beused for treatment of wounds or other acute or chronic conditions of theepidermis.

Use of an isolated compound of formula I as defined above, includingselected embodiments as described above, in the manufacture of amedicament for preventing or treating disease or condition. Use of anisolated compound of formula I as defined above, including selectedembodiments as described above, in the manufacture of a medicament forpreventing or treating disease subject to prevention or treatment byincreasing telomerase activity in a cell or tissue. Use of an isolatedcompound of formula I as defined above, including selected embodimentsas described above, for preventing or treating a disease or condition.Use of an isolated compound of formula I as defined above, includingselected embodiments as described above, for preventing or treating adisease subject to prevention or treatment by increasing telomeraseactivity in a cell or tissue The use may further comprise thepreliminary step of identifying a cell or tissue in which an increase intelomerase activity is desired. Benefits to be realized by increasingtelomerase activity in a cell or tissue include, for example,enhancement of the replicative capacity and/or life span of said cell orcells within said tissue and enhancement of functional capacity.

The use may include identifying, determining or diagnosing a conditionor disease in a subject such that increasing telomerase activity in thecells or tissue of the subject is desired. Such conditions may include,for example, viral and opportunistic infections including HIV, variousdegenerative diseases, such as neurodegenerative disease, degenerativedisease of the bones or joints and connective tissues, diabeticretinopathy, macular degeneration, cardiovascular diseases includingcentral and peripheral vascular disease, Crohn's disease and otherimmunological conditions, liver diseases including fibrosis andcirrhosis, lung disease including pulmonary fibrosis, asthma, emphysema,and COPD, hematopoietic disorders (including anemia, thrombocytopenia,neutropenia and other cytopenias), chronic inflammatory gastrointestinaldiseases such as Barretts esophagus, any disorder related to loss ofproliferative capacity in stem cell or progenitor cell populations. Suchconditions may include bone marrow failure syndrome, aplastic anemia,myelodysplastic anemia or myelodysplastic syndrome. Such conditions alsoinclude wounds or other acute or chronic conditions of the skin and itsappendages, such as, for example, a burn, an abrasion, an incision, agraft, a lesion cause by an infections agent, a chronic venous ulcer, adiabetic ulcer, a compression or decubitus ulcer, a mucosal ulcer,keloid formation, hair or pigment loss, and other structural aberrationsof the skin and its appendages. Such conditions also include cancer andprecancerous conditions in which low telomerase or shortened telomeresare associated with genomic instability, or increased mutation rates, orloss of tumor suppressor functions, and consequently subjects have anincrease risk of tumor initiation, tumor progression, or tumorrecurrence.

Similarly, use of an isolated compound of formula I, as defined above,including selected embodiments as described above, for the manufactureof a medicament for treatment of a chronic or acute condition of theepidermis is contemplated. Another embodiment is the use of an isolatedcompound of formula I, as defined above, including selected embodimentsas described above, for the treatment of a of a chronic or acutecondition of the epidermis.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an increase of telomerase activity in mice peripheral bloodmononuclear cells (PBMC) after one dose of compound 4C3-(L)-valyl-cycloastragenol, as measured in a TRAP assay.

FIG. 2 shows an increase of telomerase activity in mice whiskers afterone dose of compound 4 C3-(L)-valyl-cycloastragenol, as measured in aTRAP assay.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The following terms, as used herein, have the meanings given below,unless indicated otherwise.

A general carbon atom numbering scheme used for nomenclature ofcompounds described herein is shown below.

Thus C3-(L) valyl cycloastragenol refers to the (L) valine attachedthrough an ester bond to carbon 3 of the compound structure.

“C₁₋₅ Alkyl” refers to a fully saturated acyclic monovalent radicalcontaining carbon and hydrogen, which may be branched or linear havingfrom 1 to 5 carbon atoms. Examples of alkyl groups are methyl, ethyl,n-propyl, n-butyl, isopropyl, iso-butyl, sec-butyl, tert-butyl.

“” means ═O.

“Hydroxy” means —OH.

The term “amino acid” comprises the residues of the natural amino acids(e.g. Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as wellas unnatural amino acids. The term also comprises natural and unnaturalamino acids bearing a conventional amino protecting group (e.g. acetylor benzyloxycarbonyl). Other suitable protecting amino protecting groupsare known to those skilled in the art (See for example, T. W. Green,Protecting Groups in Organic Synthesis; Third Edition, Wiley, New York1999). Unless otherwise stated amino acid substituents are attached tothe cycloastragenol through their carboxy groups via ester linkages.Thus C3-(L) valyl cycloastragenol is C3-(L) valyl cycloastragenol ester.

The term “isomer” includes, but is not limited to optical isomers andanalogs, structural isomers and analogs, conformational isomers andanalogs and the like.

It will be appreciated by those skilled in the art that compounds of theinvention having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form or mixtures thereof, of a compound of the inventionwhich possess the useful properties described herein, it being wellknown in the art how to prepare optically active forms (for example, byresolution of the racemic form by recrystallization techniques, bysynthesis form optically-active starting materials, by chiral synthesis,or by chromatographic separation using a chiral stationary phase) andhow to determine the ability of the compounds to increase telomeraseactivity using the tests described herein. In one embodiment the aminoacids are in the naturally occurring (L) form.

The invention includes “pharmaceutically acceptable salts” of thecompounds of this invention, which may be produced, in one embodiment,to form alkali metal salts and to form free addition salts of free acidsor free bases. Suitable pharmaceutically acceptable acid addition saltsof compounds of this invention may be prepared from an inorganic acid orfrom an organic acid. In one embodiment, examples of inorganic acids arehydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric andphosphoric acid. In one embodiment, organic acids may be selected fromaliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,carboxylic and sulfonic classes of organic acids, examples of which areformic acid, acetic, propionic, succinic, glycolic, gluconic, lactic,malic, tartaric, citric, ascorbic, glucornoic, maleic, fumaric, pyruvic,aspartic, glutamic, benzoic, antranilic, oxalic, mesylic, salicyclic,stearic and galacturonic acid. In one embodiment, suitablepharmaceutically-acceptable base addition salts of compounds of thisinvention include metallic salts made from aluminum, calcium, lithium,magnesium, potassium, sodium and zinc or organic salts made fromN,N′-dibenzylethylenediamine, choline, chloroprocaine, ethanolamine,ethylenediamine, and procain. All of these salts may be prepared byconventional means from the corresponding compounds. Pharmaceuticallyacceptable salts can be prepared in other embodiments by treatment withinorganic bases, for example, sodium hydroxide. In another embodiment,esters of the compounds can be made with aliphatic and aromaticcarboxylic acids, for example, acetic acid and benzoic acid esters.

“Stem cells” refer to relatively undifferentiated cells of a commonlineage that retain the ability to divide and cycle throughout postnatallife, to provide cells that can differentiate further and becomespecialized (e.g., stem cells in basal layers of skin or inhaematopoetic tissue, such as primitive cells in the bone marrow formwhich all the various types of blood cell are derived.

By “effective to increase telomerase activity in a cell”, with referenceto a compound, is meant that a composition containing the compound at aconcentration of 10 μM or less is effective to produce a level oftelomerase activity in a keratinocyte or fibroblast cell, as measured ina telomerase activity assay (e.g. TRAP) assay as described herein, whichis greater, by a factor of at least 1.5 (i.e. at least 50% greater),than the level produced by a similar formulation not containing thecompound, as measured in a TRAP assay. In some embodiments, the compoundis effective, at a concentration of 10 μM or less, to produce a level oftelomerase activity in such a cell, as measured in a TRAP assay asdescribed herein, which is greater by a factor of at least 2 (i.e. atleast 100% greater) than the level produced by a similar formulation notcontaining the compound.

A “subject” is a mammal. The subject may be a domestic mammal forexample a dog, cat mouse, rat, monkey etc. The subject or patient may bea human.

In reference to administration of a compound to a patient, an “effectiveamount” refers to an amount effective to increase telomerase activity inthe cells or tissue of the patient, such that a desired therapeuticresult is achieved. In reference to treatment of cells in vitro or exvivo, an “effective amount” refers to an amount effective to increasetelomerase activity in the cells, thereby increasing the replicativecapacity and/or life span of the cells.

In concentrations expressed herein as % (w/v), 100% (w/v) corresponds to1 g solute/ml solvent. For example, 0.1% (w/v)=1 mg/ml.

A “formulation of an isolated compound” refers to a formulation preparedby combining the isolated compound with one or more other ingredients(which may be active or inactive ingredients) to produce theformulation. The phrase “isolated compound” refers to a compound that(prior to the formulation) has been produced by a process involving oneor more chemical synthesis steps, resulting in a preparation of thecompound that is of not less than 80% (w/w) purity.

II. Methods and Compositions for Increasing Telomerase Activity

In accordance with the present invention, compositions and methods areprovided for increasing telomerase activity in a cell.

It has been found that the compounds of the present invention are ableto increase telomerase activity in cells and are readily biologicallyavailable when administered to mammals either intravenously or orally.

In accordance with the method, a cell or tissue is contacted with anisolated compound of formula I as disclosed herein, in an amounteffective to increase telomerase activity in the cell or tissue,relative to the level of telomerase activity in the cell or tissue inthe absence of the compound. The method may also include a preliminarystep of identifying a cell or tissue in which an increase in telomeraseactivity is desired.

The present invention includes, in one aspect, a compound of the formulaI:

-   wherein X¹, is selected from keto (═O), hydroxy, and

-   wherein X² is selected from keto (═O), hydroxy, and

-   wherein X³ is selected from keto (═O), hydroxy, and

-   wherein at least one of X¹, X² or X³ is

respectively;

-   -   wherein R¹ and R² are independently selected from —CH(CH₃)₂, and        CH(CH₃)CH₂CH₃;    -   and pharmaceutically acceptable salts thereof.

In one embodiment X¹ is —OC(O)CH(NH₂)R¹ wherein R¹ is selected from thegroup consisting of —CH(CH₃)₂ or —CH(CH₃)₂CH₃ In another embodiment X²is —OC(O)CH(NH₂)R² wherein R² is selected from the group consisting of—CH(CH₃)₂ or —CH(CH₃)₂CH₃

In one embodiment at least one of X¹, X² or X³ is —OC(O)CH(NH₂)CH(CH₃)₂.In another embodiment both X¹ and X² are —OC(O)CH(NH₂)CH(CH₃)₂.

In one embodiment at least one of X¹ or X² is—OC(O)CH(NH₂)CH(CH₃)CH₂CH₃. In another embodiment both X¹ and X² are—OC(O)CH(NH₂)CH(CH₃)CH₂CH₃.

In selected embodiments of formula I, X¹ is a —OC(O)CH(NH₂)CH(CH₃)₂ andX² and X³ are independently selected from hydroxy and keto. In furtherembodiments, X² is —OC(O)CH(NH₂)CH(CH₃)₂ and X¹ and X³ are independentlyselected from hydroxy and keto. In further embodiments, X³ is—OC(O)CH(NH₂)CH(CH₃)₂ and X¹ and X² are independently selected fromhydroxy and keto. In further embodiments, X¹ and X² are both—OC(O)CH(NH₂)CH(CH₃)₂ and X³ is OH. In still further embodiments, X¹ is—OC(O)CH(NH₂)CH(CH₃)₂ and each of X² and X³ are OH. In still furtherembodiments, X² is —OC(O)CH(NH₂)CH(CH₃)₂ and each of X¹ and X³ are OH.

In selected embodiments of formula I, X¹ is a —OC(O)CH(NH₂)CH(CH₃)CH₂CH₃and X² and X³ are independently selected from hydroxy and keto. Infurther embodiments, X² is —OC(O)CH(NH₂)CH(CH₃)CH₂CH₃ and X¹ and X³ areindependently selected from hydroxyl and keto. In further embodiments,X¹ and X² are both —OC(O)CH(NH₂)CH(CH₃)CH₂CH₃ and X³ is OH. In stillfurther embodiments, X¹ is —OC(O)CH(NH₂)CH(CH₃)CH₂CH₃ and each of X² andX³ are OH. In still further embodiments, X² is—OC(O)CH(NH₂)CH(CH₃)CH₂CH₃ and each of X¹ and X³ are OH. In stillfurther embodiments, X² is —OC(O)CH(NH₂)CH(CH₃)CH₂CH₃ and each of X¹ andX³ are OH.

In some embodiments of formula I, the pharmaceutically acceptable saltis a hydrochloride salt.

Exemplary compounds of formula I include those designated herein as:2-(L)-amino-3-methyl-butyric acid6α,16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester (designated herein as 4); 2-(L)-amino-3-methyl-butyric acid6α(2-3-methyl-butyryloxy)-16β-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester (designated herein as 7);2-(L)-tert-butoxycarbonylamino-3-methyl-butyric acid3b-acetoxy-16b-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-6a-ylester (designated herein as 12) 2-(L),3-dimethyl-pentanoic acid6α,16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester (designated herein as 14); 2-(L)-Amino-3-methyl-butyric acid,16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-3-oxo-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-6α-ylester (designated herein as 30), 2-(L)-Amino-3-methyl-pentanoic acid6α-(2-amino-3-methyl-pentanoyloxy)-16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester (designated herein as 32); 2-(L)-Amino-3-methyl-butyric acid3β,6α-dihydroxy-16β-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-16β-ylester (designated herein as 36) and pharmaceutically acceptable saltsthereof.

Exemplary compounds of formulas I include those designated herein as:2-(L)-amino-3-methyl-butyric acid6α,16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester hydrochloride salt; 2-(L)-amino-3-methyl-butyric acid6α-(2-amino-3-methyl-butyryloxy)-16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester hydrochloride salt;2-(L)-tert-butoxycarbonylamino-3-methyl-butyric acid3b-acetoxy-16b-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-6a-ylester hydrochloride salt; 2-(L),3-dimethyl-pentanoic acid6α,16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester hydrochloride salt, 2-(L)-Amino-3-methyl-butyric acid,16β-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-3-oxo-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-6α-ylester hydrochloride salt; 2-(L)-Amino-3-methyl-pentanoic acid,6α-(2-amino-3-methyl-pentanoyloxy)-16β-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester hydrochloride salt or 2-(L)-Amino-3-methyl-butyric acid,3β,6α-dihydroxy-16β-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-16β-ylester hydrochloride salt.

In one embodiment, the compound is selected from the following compoundsof formula I:

and pharmaceutically acceptable salts thereof.

Exemplary compounds of formula I include the compounds in the followingtable, with reference to formula I:

Compound number Name X¹ X² X³ 4 2-(L)-amino-3-methyl-butyric acid6α,16β- —OC(O)CH —OH OH dihydroxy-17-[5-(1-hydroxy-1-methyl- (NH₂)CHethyl)-2-methyl-tetrahydro-furan-2-yl]- (CH₃)₂4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren- 3β-yl ester 72-(L)-Amino-3-methyl-butyric acid 6α-(2- —OC(O)CH —OC(O)CH —OHamino-3-methyl-butyryloxy)-16β-hydroxy- (NH₂)CH (NH₂)CH17-[5-(1-hydroxy-1-methyl-ethyl)-2- (CH₃)₂ (CH₃)₂methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]penanthren-3β-yl ester 122-(L)-tert-Butoxycarbonylamino-3-methyl- —OH —OC(O)CH —OH butyric acid3b-acetoxy-16b-hydroxy-17- (NH₂)CH[5-(1-hydroxy-1-methyl-ethyl)-2-methyl- (CH₃)₂tetrahydro-furan-2-yl]-4,4,13,14- tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-6a-yl ester 14 2-(L),3-Dimethyl-pentanoicacid 6α,16β- —OC(O)CH —OH —OH dihydroxy-17-[5-(1-hydroxy-1-methyl-(NH₂)CH ethyl)-2-methyl-tetrahydro-furan-2-yl]- (CH₃)CH₂CH₃4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenan- thren-3β-yl ester 302-(L)-Amino-3-methyl-butyric acid, 16β- ═O —OC(O)CH —OHhydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)- (NH₂)CH2-methyl-tetrahydro-furan-2-yl]-4,4,13,14- (CH₃)₂tetramethyl-3-oxo-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren- 6α-yl ester 322-(L)-Amino-3-methyl-pentanoic acid 6α- —OC(O)CH —OC(O)CH —OH(2-amino-3-methyl-pentanoyloxy)-16β- (NH₂)CH (NH₂)CHhydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)- (CH₃)CH₂CH₃ (CH₃)CH₂2-methyl-tetrahydro-furan-2-yl]-4,4,13,14- CH₃tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-yl ester 362-(L)-Amino-3-methyl-butyric acid, 3β,6α- —OH —OH —OC(O)dihydroxy-16β-hydroxy-17-[5-(1-hydroxy- CH(NH₂)1-methyl-ethyl)-2-methyl-tetrahydro-furan- CH(CH₃)₂2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren- 16β-yl ester

A compound of formula I above, when formulated in a solvent, iseffective to produce a level of telomerase activity in keratinocytes orfibroblasts, as measured in a TRAP assay, at least 50% greater, at least70% greater, at least 80% greater, or at least 90% greater than thelevel in said cells treated with said solvent, as measured in a TRAPassay as described herein. In further embodiments, the compound iseffective to produce a level of telomerase activity in keratinocytes orfibroblasts, as measured in a TRAP assay, at least 100% greater than thelevel in said cells treated with said solvent, as measured in a TRAPassay as described herein.

The invention also provides pharmaceutical compositions comprising oneor more compounds of formula I.

In a further aspect, the invention provides a method of increasingtelomerase in a cell or tissue, by contacting the cell or tissue with anisolated compound of formula I. Again, the method may include the stepof identifying a cell or tissue in which an increase in telomeraseactivity is desired.

III. Sources and Syntheses of Compounds of Formula I

The compounds of formula can be synthesized as follows.

Astragalosides I-VII can be isolated from Astragalus membranaceus root,as described, for example, in A. Kadota et al., JP Kokai No. 62012791 A2(1987). As reported therein, the root tissue (8 kg), which iscommercially available from various sources of beneficial herbs, isrefluxed with MeOH, and the concentrated extract (200 g) is redissolvedin MeOH and fractionated by column chromatography on silica gel, usingCHCl₃/MeOH/H₂O mixtures as eluants. Each fraction is worked up byreverse chromatography on silica gel, using similar solvent mixtures, togive the following approximate quantities of isolated compounds:acetylastragalosie I (0.2 g), astragaloside I (3.5 g), isoastragalosideI (0.3 g), astragaloside II (2.3 g), astragaloside III (1.0 g),astragaloside IV (0.8 g), astragaloside V (0.1 g), astragaloside VI (0.3g), and astragaloside VII (0.1 g), See also Kitagawa et al., Chem.Pharm. Bull. 31(2):698-708 (1983b).

Cycloastragenol (2) can be prepared by treatment of astragaloside IV (1)with methanolic HCl, followed by neutralization, standard workup, andpurification by chromatography, as described in the Experimental sectionbelow (Example 1). Cycloastragenol can also be obtained by oxidativedegradation (treatment with oxygen and elemental sodium) of a butanolextract of Astragalus membranaceus, as described by P-H Wang et al., J.Chinese Chem. Soc. 49:103-6 (2002).

Preparation of the various embodiments of formula I, e.g. compoundshaving varying degrees of esterification, alkylation or acylation, orketo groups, can be prepared according to known methods of organicsynthesis, using naturally occurring and/or commercially availablestarting materials such as cycloastragenol, with separation of productsas needed. Several examples are given in the Experimental section below.

IV Determination of Biological Activity

A. TRAP Assay Protocol

The ability of a compound to increase telomerase activity in a cell canbe determined using TRAP (Telomeric Repeat Amplification Protocol)assay, which is known in the art (e.g. Kim et al., U.S. Pat. No.5,629,154; Harley et al., U.S. Pat. No. 5,891,639). As used herein,“telomerase activity as measured in a TRAP assay” refers to telomeraseactivity as measured in keratinocytes or fibroblasts according to thefollowing protocol. The activity is typically compared to the activitysimilarly measured in a control assay of such cells (e.g., a telomeraseactivity 50% greater than observed in a solvent control).

Cell lines suitable for use in the assay, normal human peripheral bloodmononuclear cells (PBMCs) or Human Epidermal Keratinocytes (neonatal)(HEKs), can be obtained from commercial sources, such as CascadeBiologics, Portland, Oreg. or 4C Biotech, Seneffe, Belgium, or from theATCC (American Type Culture Collection). ATCC normal human fibroblastcell lines, which can be located on the ATCC web site, include forexample, CCL135, CCL137, and CCL151.

For example, neonatal human epidermal keratinocytes (HEKs) are platedinto a 96-well microtiter plate at approx. 5000 cells/well, in growthmedium (e.g. Epi-Life Medium+keratinocyte Growth Supplement supplied byCascade Biologics, Inc.) and incubated for one day. Test compositions ina suitable solvent, such as 95% ethanol or DMSO, are added to selectedwells in a range of concentrations and incubated for a further 24+/−1hours.

Compounds to be tested are first formulated at a 10× desired finalconcentration in 10% DMSO. The formulated compound is added to the96-well culture along with a control of DMSO to provide variousconcentrations of the compound. The final DMSO concentration may be 1%in all wells. For other cell types or in other situations, higher orlower concentrations of DMSO may be desired.

A cytotoxicity assay may be performed in parallel with the telomeraseTRAP testing by preparing a duplicate cell culture plate treated withthe same compounds and using a metabolism responsive dye such as AlamarBlue to assess the number of cells at the beginning and the end of theincubations with the test compounds.

If cytotoxicity of the test compounds is not objectively measured, themorphology of treated cells can first be observed under a microscope, toverify that there are no visual signs of irregular growth.

To conduct the TARP assay, media is removed from the wells, and thecells are rinsed twice in PBS (Ca and Mg free). The dishes are chilledon ice, and Nonidet P40 cell lysis buffer is added (approx. 100 μl perwell) and triturated by pipetting up and down several times. The cellsare the incubated on ice for 1 hour.

Alternatively, cells may be harvested at 24 hr+/−1 hr by removing thegrowth medium and washing once with PBS (phosphate buffered saline)removing as much medium as possible. The cells are then lysed by adding50 μL of M-Per buffer (Pierce Cat#78503 & 78501) and incubating on icefor 1 hr+/−15 min. The plate is, optionally, centrifuged at 2000 RPM, 5min. The lysate is carefully collected from each well of the plate andtransferred to a fresh V-bottom storage 96-well plate, leaving themonolayer cells intact.

Alternatively, cell lysing solution may be prepared by addition of 3.0mL Nonidet® P40, 1.0 mL CHAPS lysis buffer (see below), and 1.0 mL 10×TRAP buffer (see below) to 5.0 mL DNase-, RNase-free H₂O. (DNase-,RNase-free water may be generated by DEPC (diethylpyrocarbonate)treatment or purchased from vendors such as Sigma).

CHAPS Lysis Buffer Stock For 1 mL Final concn.   1M Tris-HCl pH 7.5 10μl 10 mM   1M MgCl₂ 1 μl 1 mM 0.5M EGTA 2 μl 1 mM 100 mM AEBSF 1 μl 0.1mM  10% CHAPS^(a) 50 μl 0.5% BSA 1 mg 1 mg/ml 100% Glycerol 100 μl 10%DNase-, RNase-free H₂O 936 μl (to 1 mL) ^(a)The CHAPS detergent is addedjust before use of the lysis buffer. In addition, AEBSF(4-(2-aminoethyl)-benzenesulfonyl fluoride HCl) is added to the lysisbuffer just prior to the extraction step.

The level of telomerase activity in the cell lysates is measured using aTRAP assay.

10X TRAP Buffer Stock Final concn.   1M Tris-HCl, pH 8.3 200 mM   1MMgCl₂  15 mM   1M KCl 650 mM Tween 20 (Boehringer Mannheim) 0.5% 0.1MEGTA  10 mM 20 mg/ml BSA  1 mg/ml

The following materials are combined to generate a master PCR Mix.

Stock Per Reaction (45 μl) Final concn.^(a) 10X TRAP Buffer 5.0 μL 1X2.5 mM dNTPs 1.0 μL 50 μM Cy5-TS Primer (0.5 mg/ml) 0.1 μL 1 ng/ml ACXPrimer (0.1 mg/ml) 1.0 μL 2 ng/ml Taq Polymerase (5 U/μl) 0.4 μL 0.04units/μl Cell extract 5-10 μL DNase-, RNase-free H₂O 32.5-37.5 μL (to 45μL total) ^(a)Based on final volume of 40 μl PCR mix plus 10 μl celllysate = 50 μl.

The PCR mix includes the following components: Cy5-TS primer, a 5′-Cy5labeled oligonucleotide having the sequence 5′-AAT CCG TCG AGC AGAGTT-3′ (SEQ ID NO:1), is a telomerase substrate. Depending on thetelomerase activity in the medium, telomere repeats (having the sequence(AGGGTT)_(n) will be added to the substrate, to form telomerase extendedproducts, also referred to as telomerase products. The ACX primer,having the sequence 5′-GCG CGG CTT ACC CTT ACC CTT ACC CTA ACC-3′ (SEQID NO:2), is an anchored return primer that hybridizes to the telomeraseextended products.

A sample of cell lysate (e.g., 5 μL) is added to the PCR mix in areaction tube, and the telomere extension and PCR amplification is donein the bench top PCR machine at the following cycle profiles: 30° C. for30 minutes, repeat 28 cycles of the following 3 step reaction: 94° C./30sec, 60° C./30 sec, and 72° C./1 min, followed by 72° C./4 minutes andhold at 4° C.

Loading dye containing e.g. bromophenol blue and xylene cyanol is added,and the samples are subjected to 10-15% non-denaturing PAGE in 1×TBE,until the bromophenol blue runs off the gel. The TRAP reaction productis observed, e.g. by using a fluoroimager for detection of CY5-labeledtelomerase products (maximal excitation at 650 nm; maximal emission at670 nm).

Telomerase activity may be measured by captured total pixel vol. (DNAladder bands) above background for each gel lane. The activity may benormalized by measuring the total RNA (ng/mL) by using Ribogreen® RNAQuantitation Kit from Molecular Probes, cat. # R-11490 and followingcommercially recommended conditions with an RNA standard range of0.8-200 ng/mL, 1:200 dilution of RG dye, 100-250× dilution of sample.

Total Pixel Vol/RNA=Normalized Relative Telomerase Activity

-   Cells number (used to assess cytotoxicity) was directly proportional    to the Alamar Blue reading

Alternatively, a set of an internal standard and primer can be added forquantitation purposes. The TSU2 internal standard an oligonucleotidehaving the sequence 5′-AAT CCG TCG AGC AGA GTT AAA AGG CCG AGA AGCGAT-3′; SEQ ID NO:3), an extension of the TS primer sequence, is addedin a small controlled quantity. The U2 primer, having the sequence5′-ATC GCT TCT CGG CCT TTT (SEQ ID NO:4), is a return primer designed tohybridize to the 3′ region of the internal standard.

The final amount of TSU2 internal standard after amplification isgenerally 5-10 pmol per 50 μl reaction mixture. This internal controlgives a specific 36-mer PCR amplification product that appears as adistinct band on the gel below the first telomere addition product thatappears as a distinct band on the gel below the first telomere additionproduct (that is, the product of one telomer addition to the TSoligonucleotide, followed by amplification with the ACX return primer).This internal control band can be used to normalize the PCRamplifications from different samples.

The relative number of telomerase product molecules (TM) generated inthe assay is determined according to the formula below:

TM=(T _(TRAP Products) −T _(BKD1))/(T _(Int Std) −T _(BKD2))

where: T_(TRAP Products) is the total intensity measured on the gel forall telomerase products, T_(BKD1) is the background intensity measuredin a blank lane for an area equivalent in size to that encompassed bythe telomerase products, T_(Int Std) is the intensity for the internalstandard band, and T_(BKD2) is the background intensity measured in ablank lane for an area equivalent in size to that encompassed by theinternal standard band. The resulting number is the number of moleculesof telomerase products generated for a given incubation time, which, forthe purposes of determining TM, is designated herein as 30 minutes.

Compounds of formulas I as described above are able to produce, at aconcentration of 1 μM or less, a level of telomerase activity infibroblasts or keratinocytes at least 50% greater than seen in a solventcontrol. Even more potent activities may be appropriate for someapplications, such as compounds that produce telomerase activities atleast about 75%, 100% or 500% greater than the level of such activityseen in a solvent control, as measured in the described TRAP assay, at aconcentration of 10 μM or less.

Effectiveness in increasing telomerase activity was evaluated forcompounds of formula I above in various concentrations. Assays werecarried out in HEKneoP cells (neonatal keratinocytes), according to theprotocol described above. Concentrations typically ranged from approx.0.001 μM to 10 μM in DMSO.

The ability of the compounds to increase the activity of telomerase asshown in Table 2.

B. Wound Healing Assay Protocol

The compounds of formula I can be used to promote healing of wounds,burns, abrasions or other acute or chronic conditions of the epidermis,as discussed further below. As used herein, “wound healing activity asmeasured in a scratch assay” refers to the activity as measured inkeratinocytes or fibroblasts according to the following protocol, andexpressed as the value of WH shown in the formula below.

Cells are plated in flasks (5×10⁵ cells per flask) and cultured for twodays in a humidified chamber at 5% CO₂, 37° C. To create the “wound”, a2 ml plastic pipette is gently dragged to “scratch” the cell surface.The ideal wound is approximately 2-3 mm wide and 50 mm long (along thelong axis of the tissue culture flask). The cells are retreated withmedium containing either vehicle (DMSO; control sample) or testcompositions at multiple concentrations. A wound area is identified, theflask marked, and the appearance of the cells documentedphotographically over 3-4 days continued culturing of the cells.

Amount of wound closure is determined by measuring the width of thewound over time for compound-treated samples relative to vehicle-treatedor other control cells. Measurements are made from the photographs takenfor each of the samples on days 1 (immediately after scratching), 2, 3,and 4. Percentage of wound healing (also expressed as “wound healingactivity”) is calculated by the following formula:

WH=100−[100×W _(n) /W ₀],

where W_(n) is the width of the wound on day n and W₀ is the width ofthe wound on day one (i.e. immediately after scratching).

V. Therapeutic Indications and Treatment Methods

The present invention provides methods for increasing telomeraseactivity in a cell, by contacting a cell or tissue with a formulation ofan isolated compound of formula I as disclosed in Section II above, inan amount effective to increase telomerase activity in the cell. Themethod may include the preliminary step of identifying a cell or tissuein which an increase telomerase activity is desired. The cell may be inculture, i.e. in vitro or ex vivo, or within a subject or patient invivo.

Benefits to be realized from an increase in telomerase activity in acell or tissue include, for example, enhancement of the replicativecapacity and/or life span of the contacted cells and improved functionalcapacity of the cells (i.e. improved expression of the normaldifferentiated functions of the cells). The method may further comprisediagnosing a condition in a subject or patient wherein an increase intelomerase activity in cells or tissue of the patient is desired; e.g.,diagnosing a disease subject to treatment by an increase in telomeraseactivity in cells or tissue. Accordingly, the invention provides methodsof treating a condition in a patient, by increasing telomerase activityin cells or tissue of said patient, the method comprising administeringto a subject in need of such treatment an effective amount of a compoundof formula I as disclosed in Section II above. An “effective amount”refers to an amount effective to increase telomerase activity in thecells or tissue of the patient, such that a therapeutic result isachieved.

Such conditions or diseases for treatment or prevention may include, forexample, conditions associated with cellular senescence or with anincreased rate of proliferation of a cell in the absence of telomerase,which leads to accelerated telomere repeat loss. By “increased rate ofproliferation” is meant a higher rate of cell division compared tonormal cells of that cell type, or compared to normal cells within otherindividuals of that cell type. The senescence of those groups of cellsat an abnormally early age can eventually lead to disease (see West etal., U.S. Pat. No. 6,007,989).

Various disease states exist in which an increase in telomerase activityin certain cell types can be beneficial. Accordingly, the inventionprovides methods of treating in a patient a condition or diseaseselected from the following, by increasing telomerase activity in thecells of the patient, comprising administering to a subject in need ofsuch treatment, an effective amount of a compound of formula I asdescribed above. In some cases, the condition may also be subjected totreatment by ex vivo cell therapy, as described further below, employingthe associated cell types (indicated in parenthesis).

(a) Alzheimer's disease, Parkinson's disease, Huntington's disease, andstroke (cells of the central nervous system, including neurons, glialcells, e.g. astrocytes, endothelial cells, fibroblasts).

(b) age-related diseases of the skin, such as dermal atrophy andthinning, elastolysis and skin wrinkling, sebaceous gland hyperplasia orhypoplasia, senile lentigo and other pigmentation abnormalities, grayingof hair and hair loss or thinning, or chronic skin ulcers (fibroblasts,sebaceous gland cells, melanocytes, keratinocytes, Langerhan's cells,microvascular endothelial cells, hair follicle cells),

(c) degenerative joint disease (cells of the articular cartilage, suchas chrondrocytes and lacunal and synovial fibroblasts),

(d) osteoporosis and other degenerative conditions of the skeletalsystem (cells of the skeletal system, such as osteoblasts, bone marrowstromal or mesenchymal cells, osteoprogenitor cells),

(e) age- and stress-related diseases of the vascular system includingatherosclerosis, calcification, thrombosis, and aneurysms (cells of theheart and vascular system, including endothelial cells, smooth musclecells, and adventitial fibroblasts),

(f) age-related macular degeneration (cells of the eye, such aspigmented epithelium and vascular endothelial cells),

(g) AIDS (HIV-restricted CD8⁺ cells);

(h) age- and stress-related immune system impairment, includingimpairment of tissue turnover, which occurs with natural aging, cancer,cancer therapy, acute or chronic infections, or with genetic disorderscausing accelerated cell turnover, and related anemias and otherdegenerative conditions (other cells of the immune system, includingcells in the lymphoid, myeloid, and erythroid lineages, such as B and Tlymphocytes, monocytes, circulating and specialized tissue macrophages,neutrophils, eosinophils, basophils, NK cells, and their respectiveprogenitors); and

(i) pulmonary fibrosis or liver cirrhosis or liver fibrosis;

(j) chronic inflammatory gastrointestinal diseases such as Barrettsesophagus; and

(k), bone marrow failure syndrome, aplastic anemia, myelodysplasticanemia or myelodysplastic syndrome.

In addition to the cell types noted above, further cell types in whichan increase in telomerase activity can be therapeutically beneficialinclude, but are not limited to, cells of the liver, endocrine andexocrine glands, smooth musculature, or skeletal musculature.

As an example, in the case of HIV-infected individuals, CD8⁺ cellturnover is increased as these cells attempt to control the level ofHIV-infected CD4⁺ cells. In AIDS (item (g) above), disease is believedto be caused by the early senescence of HIV-restricted CD8⁺ cells. Theaging of such cells is attributed not simply to abnormal amount of lossof telomere sequences per cell doubling, but, in addition, to theincreased replicative rate of the cells, such that telomere attrition isgreater than normal for that group of cells. The invention thus providesmethods of treating an HIV infected subject, and more particularly ofreducing early sensecence of HIV-restricted CD8⁺ cells in an HIVinfected subject, by administering to a subject in need of suchtreatment an effective amount of a compound of formula I as disclosed inSection II above.

An increase in telomerase activity can benefit non-dividing cells aswell as proliferating cells, e.g. in conditions associated withincreased susceptibility to cell death due to stress, such as ischemiain heart failure or in stroke (see e.g. Oh and Schneider, JMol CellCardiol 34(7):717-24; Mattson, Exp Gerontol. 35(r):489-502). Theinvention thus provides methods of reducing stress- orDNA-damage-induced cell death in a subject, such as a subjectexperiencing ischemic conditions in tissue due to heart failure. orstroke, by increasing telomerase activity in cells of the subject,comprising administering to a subject in need of such treatment aneffective amount of a compound of formula I as disclosed in Section IIabove. As noted above, the method may include the preliminary step ofdiagnosing in the subject the indicated condition.

In another aspect, the compositions may be used for the treatment ofindividuals in which one or more cell types are limiting in thatpatient, and whose life can be extended by extending the ability ofthose cells to continue replication or resist stress-induced cell death.One example of such a group of cells is lymphocytes present in Down'sSyndrome patients. The invention thus provides a method of enhancingreplicative capacity and/or life span of lymphocytes present in Down'sSyndrome patient, by increasing telomerase activity in said cells of thepatient, comprising administering to such a patient an effective amountof a compound of formula I as disclosed in Section II above. Thecompositions may also be used to improve resistance to stress-inducedcell death occurring during normal aging.

In a further aspect of the invention, increasing telomerase activity iseffective to prevent pulmonary fibrosis or to promote healing ofpulmonary fibrosis. It has been determined that short telomeres are asignature of idiopathic pulmonary fibrosis and of cryptogenic livercirrhosis (Alder et al., PNAS (2008) 105(35) 13051-13056). The presentcompounds may be used to treat pulmonary fibrosis or liver cirrhosis.

In a further aspect, the invention provides a method of enhancingtransplantation of a tissue from a living donor or cadaver to a livingpatient or subject comprising contacting the transplantation tissue withan isolated compound of formula I as defined above. In a further aspect,the invention provides a method of enhancing transplantation of a tissueto a living patient or subject comprising administering the isolatedcompound of formula I as defined above to the patient either before,simultaneous with, or for a period of time after the transplantation ofthe tissue into the patient. The transplanted tissue may be solidtissue, such as a kidney, heart, lungs etc., or hematopoietic tissuesuch as, without limitation, blood cells such as leukocytes, lymphocytesor hematopoietic precursor cells which may be derived from bone marrow.

In a further aspect of the invention, increasing telomerase activity iseffective to promote healing of wounds, burns, abrasions or other acuteor chronic conditions of the epidermis. The invention thus provides amethod of treating an acute or chronic condition of the epidermis, byadministering to a patient in need of such treatment, topically to theaffected area, an effective amount of a formulation of an isolatedcompound of formula I as disclosed in Section II above.

As used herein, an “acute or chronic condition of the epidermis”includes acute conditions such as lesions suffered in trauma, burns,abrasions, surgical incisions, donor graft sites, and lesions caused byinfections agents, and chronic conditions such as chronic venous ulcer,diabetic ulcer, compression ulcer, pressure sores, and ulcers or soresof the mucosal surface. Also included are skin or epithelial surfacelesions caused by a persistent inflammatory condition or infection, orby a genetic defect (such as keloid formation and coagulationabnormalities). See, for example, PCT Pubn. No. WO 02/91999.

Desirable effects of an increase in telomerase activity in suchtreatment include cell proliferation or migration at the treatment site,epithelialization of the surface, closure of a wound if present, orrestoration of normal physiological function. By “epithelialization” or“reepithelialization” of a treatment site is meant an increase indensity of epithelial cells at the site as a result of the appliedtherapy.

The method may also be used to enhance growth of engrafted cells.Desirable effects of an increase in telomerase activity in suchtreatment include coverage of the treatment site, survival of engraftedcells, lack of immune rejection, closure of a wound if present, orrestoration of normal physiological function. Engrafted cells mayparticipate in wound closure either by participating directly in thehealing process (for example, becoming part of the healed tissue), or bycovering the wound and thereby providing an environment that promoteshealing by host cells.

The invention also contemplates manipulation of the skin and repair ofany perceived defects in the skin surface.

In a further aspect, the methods and compositions of the invention canbe used to enhance replicative capacity and/or extend life span of cellsin culture, e.g. in ex vivo or in vitro cell therapy or in monoclonalantibody production, by increasing telomerase activity in the cells.Increasing telomerase activity increases the replicative capacity ofsuch cells by slowing telomere repeat loss and/or improving resistanceto stress-induced cell death during cell proliferation.

In the case of ex vivo application, an effective amount of a compound offormula I as described above is added to explant cells obtained from asubject. An “effective amount” refers to an amount effective to increasetelomerase activity in the cells, thereby increasing the replicativecapacity and/or life span of the cells.

The explant cells may include, for example, stem cells, such as bonemarrow stem cells (U.S. Pat. No. 6,007,989), bone marrow stromal cells(Simonsen et al., Nat Biotechnol 20(6):592-6, 2002), or adrenocorticalcells (Thomas et al, Nat Biotechnol 18(1):39-42, 2000). Diseaseconditions such as those noted in items (a)-(g) above may also besubject to ex vivo cell-based therapy. Examples include the use ofmuscle satellite cells for treatment of muscular dystrophy, osteoblaststo treat osteoporosis, retinal pigmented epithelial cells forage-related macular-degeneration, chondrocytes for osteoarthritis, andso on.

For example, the recognition that functional CD8⁺ cells are limiting inAIDS patients to control the expansion of infected CD4+ cells allows atherapeutic protocol to be devised in which HIV-restricted CD8⁺ cellsare removed from an HIV-infected individual at an early stage, when AIDSis first detected, stored in a bank, and then reintroduced into theindividual at a later stage, when that individual no longer has therequired CD8⁺ cells available. Thus, an individual's life can beextended by a protocol involving continued administration of thatindividual's limiting cells at appropriate time points. Theseappropriate points can be determined by following CD8⁺ cell senescence,or by determining the length of telomeres within such CD8⁺ cells, as anindication of when those cells will become senescent. In accordance withthe invention, the stored cells can be expanded in number in thepresence of an agent which slows telomere repeat loss, i.e. compound offormula I as disclosed in Section II above.

Accordingly, the invention provides methods of ex vivo cell basedtherapy, which include obtaining a cell population from a subject, andexpanding the cell population ex vivo, wherein the cell population istreated with a compound of formula I as disclosed in Section II above,in an amount effective to increase telomerase activity and therebyenhance the replicative capacity and/or life span of the cellpopulation. The method generally includes diagnosing in a subject acondition subject to a treatment by ex vivo cell based therapy, such asthose noted above.

In a further embodiment, the invention provides a method of stem cellproliferation, wherein a stem cell population is treated with a compoundof formula I as disclosed in Section II above, in an amount effective toincrease telomerase activity and thereby enhance the replicativecapacity and/or life span of the cell population.

VI. Formulations and Methods of Administration

The invention encompasses methods of preparing pharmaceuticalcompositions useful for increasing telomerase activity in a cell and/orpromoting wound healing. Accordingly, an isolated compound of formula Ias described in Section II is combined with a pharmaceutical excipient,and optionally with other medicinal agents, adjuvants, and the like,which may include active and inactive ingredients. The compositions maytake the form of solid, semi-solid, lyophilized powder, or liquid dosageforms, such as, for example, tablets, capsules, powders,sustained-release formulations, solutions, suspensions, emulsions,suppositories, creams, ointments, lotions, aerosols, or the like. Theformulations may be provided in unit dosage forms suitable for simpleadministration of precise dosages.

An isolated compound of formula I may also be formulated for oraladministration. For an oral pharmaceutical formulation, suitableexcipients include pharmaceutical grades of carriers such as mannitol,lactose, glucose, sucrose, starch, cellulose, gelatin, magnesiumstearate, sodium saccharine, and/or magnesium carbonate. For use in oralliquid formulations, the composition may be prepared as a solution,suspension, emulsion, or syrup, being supplied either in solid or liquidform suitable for hydration in an aqueous carrier, such as, for example,aqueous saline, aqueous dextrose, glycerol, or ethanol, polyethyleneglycol, macrogol-15 hydroxystearate or for example water or normalsaline. If desired, the composition may also contain minor amounts ofnon-toxic auxiliary substances such as wetting agents, emulsiyfingagents, or buffers.

For use in wound healing or treatment of other acute or chronicconditions of the epidermis, a compound of formula I is formulated fortopical administration. The vehicle for topical application may be inone of various forms, e.g. a lotion, cream, gel, ointment, stick, spray,or paste. These product forms can be formulated according to well knownmethods. They may comprise various types of carriers, including, but notlimited to, solutions, aerosols, emulsions, gels, and liposomes. Thecarrier may be formulated, for example, as an emulsion, having anoil-in-water or water-in-oil base. Suitable hydrophobic (oily)components employed in emulsions include, for example, vegetable oils,animal fats and oils, synthetic hydrocarbons, and esters and alcoholsthereof, including polyesters, as well as organopolysiloxane oils. Suchemulsions also include an emulsifier and/or surfactant, e.g. a nonionicsurfactant, such as well known in the art, to disperse and suspend thediscontinuous phase within the continuous phase.

The topical formulation typically contains one or more componentsselected from a structuring agent, a thickener or gelling agent, and anemollient or lubricant. Frequently employed structuring agents includelong chain alcohols, such as stearyl alcohol, and glyceryl ethers oresters and oligo(ethylene oxide) ethers or esters thereof. Thickenersand gelling agents include, for example, polymers of acrylic ormethacrylic acid and esters thereof, polyacrylamides, and naturallyoccurring thickeners such as agar, carrageenan, gelatin, and guar gum.Examples of emollients include triglyceride esters, fatty acid estersand amides, waxes such as beeswax, spermaceti, or carnauba wax,phospholipids such as lecithin, and sterols and fatty acid estersthereof. The topical formulations may further include other componentsas known in the art, e.g. astringents, fragrances, pigments, skinpenetration enhancing agents, sunscreens, etc.

The pharmaceutical compositions may also be formulated foradministration parenterally, transdermally, or by inhalation. Aninjectable composition for parenteral administration typically containsthe active compound in a suitable IV solution, such as sterilephysiological saline. The composition may also be formulated as asuspension in a lipid or phospholipid, in a liposomal suspension, or inan aqueous emulsion.

For administration by inhalation, the active compound is formulated assolid or liquid aerosol particles. The formulation may also include apropellant and/or a dispersant, such as lactose, to facilitate aerosolformation. For transdermal administration, the active compound isincluded in a transdermal patch, which allows for slow delivery of acompound to a selected skin region, and which may also includepermeation enhancing substances, such as aliphatic alcohols or glycerol.

Methods for preparing such formulations are known or will be apparent tothose skilled in the art; for example, see Remington's PharmaceuticalSciences (19th Ed., Williams & Wilkins, 1995). The composition to beadministered will contain a quantity of the selected compound in apharmaceutically safe and effective amount for increasing telomeraseactivity in the target cells or tissue.

The pharmaceutical composition contains at least 0.1% (w/v) of acompound of formula I as described above, greater than 0.1%, up to about10%, up to about 5%, and up to about 1% (w/v). Choice of a suitableconcentration depends on factors such as the desired dose, frequency andmethod of delivery of the active agent.

For treatment of a subject or patient, such as a mammal or a humanpatient, dosages are determined based on factors such as the weight andoverall health of the subject, the condition treated, severity ofsymptoms, etc. Dosages and concentrations are determined to produce thedesired benefit while avoiding any undesirable side effects. Typicaldosages of the subject compounds are in the range of 1-50 mg/kg/day,1-25 mg/kg/day, 1-20 mg/kg/day, 4-15 mg/kg/day. Typical dosages of thesubject compounds are in the range of about 1 to 1,500 mg/day for ahuman patient, about 1-500 mg/day. In specific embodiments, for example,the compound designated herein as 4 is administered at a level of atleast 1 mg/kg/day or at least 5 mg/kg/day.

Administration of the compounds of Formula I may be every other day, ona daily basis, twice daily or more often. Administration may be once,for 1-20 days, for 5-10 days or continuously for as long as necessary toprevent or treat the disease or condition being prevented or treated.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Conversion of Astragaloside IV (1) to17-[5-(1-Hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthrene-3β,6α,16β-triol[cycloastragenol] (2)

To astragaloside IV (1) (5.00 g, mmol) was added “HCl-MeOH 10” (TCIAmerica) (500 mL) and the mixture was stirred at room temperature for 7days. The reaction mixture was concentrated to about half volume underreduced pressure at 20° C. (do not heat). The mixture was partitionedinto aqueous sodium bicarbonate and ethyl acetate. The aqueous layer wasextracted with ethyl acetate again. The organic layers were combined,washed with saturated sodium chloride, dried on anhydrous sodiumsulfate, and concentrated under reduced pressure. The residue waspurified by column chromatography (20:1˜14:1 chloroform/methanol). Inorder to replace the residual solvent with ethanol, the purifiedmaterial was dissolved in ethanol and the solvent was removed underreduced pressure to afford 2 (2.1 g, 64%).

¹H NMR (CDCl₃) δ(ppm) 0.34 (d, J=4.7 Hz, 1H), 0.48 (d, J=4.3 Hz, 1H),0.92 (s, 3H), 0.93 (s, 3H), 1.0-1.8 (m, 13H), 1.11 (s, 3H), 1.19 (s,3H), 1.22 (s, 6H), 1.27 (s, 3H), 1.9-2.0 (m, 4H), 2.30 (d, J=7.8 Hz,1H), 2.54 (q, J=11.8 Hz, 1H), 3.27 (m, 1H), 3.50 (m, 1H), 3.72 (t, J=7.4Hz, 1H), 4.65 (q, J=7.4 Hz, 1H). ESI-MS m/z Positive 491 (M+H)⁺,Negative 549 (M+AcO)⁻. TLC (Merck, Kieselgel 60) Rf=0.33 (6:1chloroform/methanol)

Example 2 Preparation of 2-(L)-Amino-3-methyl-butyric acid6α,16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester, hydrochloride salt [C3-(L)-valyl-cycloastragenol](4)

Preparation of 3: Boc-L-Valine-OH (18 g, 81.63 mmols) (Bachem, Torrance,Calif.) was dissolved in 150 ml of dichloromethane (DCM). To this wasadded 15 g (81.63 mmol) of pentafluorophenol. The reaction was cooled inan ice-bath followed by slow addition of 12.8 ml (81.63 mmols) of1,3-diisopropylcarbodiimide (DIC). After complete addition the reactionmixture was stirred at room temperature for 30 minutes at which time thereaction mixture turned turbid (diisopropylcabodimide-ureaprecipitation). To this mixture was then added 10 g (20.41 mmols) of (2)followed by 10 g (81.63 mmol) of dimethylaminopyridine (DMAP) and thereaction was stirred at room temperature for 24 hours. The reactionmixture was transferred into a sepratory funnel and washed with H₂O (2×)1% aq. HCl (2×), 0.1 N aq. NaOH (2×), sat. NaHCO₃ (3×), H₂O (1×) andbrine (1×), the organic layer was separated, dried over Na₂SO₄, filteredand the solvent was evaporated under vacuum. The residue was purifiedusing flash chromatography with solvent gradient of 2%-5% MeOH in DCM tofurnish 7.0 g (50%) of the target product 3 together with 6.0 g, (33%)of this bis product.

¹HNMR for 3: (CDCl₃) δ(ppm) 0.38 (1H, bs), 0.90-1.38 (m, 30H), 1.39-1.45(s,m 12H), 1.59-1.63 (m, 5H), 1.76-1.82 (m, 2H), 1.96-2.01 (m, 4H),2.16-2.20 (m, 1H), 2.30-2.35 (d, 1H), 2.49-2.54 (q, 1H), 3.45-3.57 (t,1H), 3.71-3.76 (t, 1H), 4.19-4.21 (m, 1H), 4.53-4.61 (m, 1H), 4.69-4.71(q, 1H), 5.0-5.2 (d, 1H. MS (M+H) 690.

Preparation of 4: to 1 g (1.45 mmol) of 3 was added 1.8 ml of 4.0 MHCl/dioxane and stirred for 4 hrs. The solvents were evaporated and theproduct was precipitated in 10 ml of cold diethyl ether and the solidswere filtered. The solids were then dried under high vacuum forovernight to yield 800 mg (88%) of the target product 42-(L)-Amino-3-methyl-butyric acid6α,16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester, hydrochloride salt as a white powder.

¹HNMR for 4 (DMSOd₆) δ(ppm): 0.36 (bs, 1H), 0.49 (bs, 1H), 0.80-1.39 (m,29H), 1.44-1.60 (m, 3H), 1.61-1.70 (m, 2H), 1.81-1.89 (m, 4H), 1.81-1.89(m, 4H), 2.19-2.30 (m, 2H), 2.41-2.60 (m, 2H), 3.29-3.41 (m, 2H),3.58-3.61 (t, 1H), 3.81-3.83 (m, 1H), 4.18-4.39 (bs, 4H), 4.49-4.51 (q,2H), 4.54-4.59 (m, 1H), 8.40-8.58 (bs, 2H), MS (M+H) 590.

Example 3 Preparation of 2-(D)-Amino-3-methyl-butyric acid6α,16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester, hydrochloride salt hydrochloride salt[C3-(D)-valyl-cycloastragenol](5)

Using the procedure of Example 2 with Boc-(D) Valine-OH (Bachem,Torrance, Calif.) 18 g, 18.63 mmols) compound 5 was prepared.

¹HNMR for 5 (DMSOd₆) δppm: 0.30 (bs, 1H), 0.50 (bs, 1H), 0.80-1.39 (m,29H), 1.46-1.58 (m, 3H), 1.61-1.70 (m, 2H), 1.79-1.89 (m, 4H), 2.16-2.32(m, 2H), 2.38-2.54 (m, 2H), 3.29-3.41 (m, 2H), 3.58-3.61 (t, 1H),3.81-3.83 (m, 1H), 4.13-4.24 (bs, 4H), 4.50-4.52 (q, 2H), 4.54-4.59 (m,1H), 8.43-8.60 (bs, 2H). MS (M+H) 590.

Example 4 Preparation of 2-(L)-Amino-3-methyl-butyric acid6α-(2-amino-3-methyl-butyryloxy)-16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester, hydrochloride salt [C3,C6-(L,L)-bisvalyl-cycloastragenol]-7

Preparation of 6: Boc-(L) Valine-OH (10 g, 46.08 mmols) was dissolved in80 ml of N-methylpyrrolidone (NMP). To this was added 8.5 g (46.08 mmol)of pentathuorophenol. The reaction was cooled in an ice-bath followed byslow addition of 7.2 ml (46.08 mmols) of DIC. After complete additionthe reaction mixture was stirred at room temperature for 30 minutes atwhich time the reaction mixture turned turbid(diisopropylcarbodimide-urea precipitation). To this mixture was thenadded 3.2 g (6.60 mmols) of 2 followed by 5.5 g (45 mmol) of DMAP andthe reaction was stirred at room temperature for 24 hours. The reactionmixture was transferred into a sepratory funnel and washed successivelywith H₂O (6×), 1% aq. HCl (2×), 0.1 N aq. NaOH (2×), sat. NaHCO₃ (3×),H₂O (1×), and brine (1×), the organic layer was separated, dried overNa₂SO₄, filtered and the solvent was evaporated under vacuum. Theresidue was purified using flash chromatography with solvent gradient of2%-5% MeOH in DCM to furnish 4.8 g (83%) of the traget product 6.

¹HNMR for 6: (CDCl₃) δppm: 0.38 (1H, bs), 0.60 (1H, bs), 0.80-1.0 (m,24H), 1.15 (s,s, 6H), 120 (s,s 6H), 1.31 (s, 6H), 1.35 (s,s, 4H) 1.41(s,s, 18H), 1.56-1.60 (m, 4H), 1.79-1.83 (m, 3H), 3.71-3.76 (t, 1H),4.08-4.21 (m, 2H), 4.58-4.60 (m, 1H), 4.61-4.70 (q, 1H), 4.72-4.80 (m,1H), 4.82-4.84 (d, 1H), 4.9-5.0 (d, 1H). MS (M+H) 889.

Preparation of 7: To a 4.5 g (5.06 mmol) of the 6 was added 13 ml of4.0M HCl/dioxane and stirred for 4 hrs. The solvents were evaporated andthe product was precipitated with 40 ml of cold diethyl ether and thesolids filtered off. The solids were then dried under high vacuum forovernight to yield 3.1 g (91%) of the target product 7 as a whitepowder.

¹HNMR for 7 (DMSOd₆) δppm: 0.24 (bs, 1H), 0.80-1.20 (m, 35H), 1.41-1.85(m, 12H), 2.10-2.22 (m, 2H), 2.32-2.42 (m, 4H), 2.19-2.30 (m, 2H),3.59-3.62 (m, 1H), 3.81-3.83 (m, 2H), 4.40-4.53 (m, 1H), 4.60-4.71 (m,1H), 4.81-4.9 (m, 1H), 8.40-8.70 (d, 4H). MS (M+H) 689.

Example 5 Preparation of 2-(D)-Amino-3-methyl-butyric acid6α-(2-amino-3-methyl-butyryloxy)-16β-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester, hydrochloride salt [C3,C6-(D,D)-bisvalyl-cycloastragenol]8

Using the same procedure of Example 4 and Boc-(D) Valine-OH (Bachem,Torrance, Calif.), compound 8 was prepared.

¹HNMR for 8 (DMSOd₆) δppm: 0.26 (bs, 1H), 0.60 (bs, 1H), 0.78-1.23 (m,35H), 1.39-1.80 (m, 12H), 2.10-2.22 (m, 2H), 2.19-2.30 (m, 2H),2.35-2.40 (m, 4H), 3.60-3.62 (m, 1H), 3.80-3.85 (m, 2H), 4.42-4.53 (m,1H), 4.58-4.70 (m, 1H), 4.81-4.9 (m, 1H), 8.40-8.70 (d, 4H). MS (M+H)689.

Example 6 Preparation of 2-(L)-tert-Butoxycarbonylamino-3-methyl-butyricacid3b-acetoxy-16b-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-6a-ylester hydrochloride salt [C6-(L)-valyl-cycloastragenol]12

Preparation of 9: To a 5 g (10.22 mmol) of 2 was added 40 ml of CHCl₃and 2.1 ml (26 mmol) of pyridine. The reaction mixture was cooled in anice-bath and to this was slowly added 2.5 ml (26 mmol) of aceticanhydride. After complete addition the reaction was stirred at 4° C. for24 hrs. The TLC showed three spots corresponding to 9, monoacetylatedand bis acetylated products. The reaction mixture was diluted with 100ml of DCM and washed successively with the following: sat. aq. NaHCO₃,(2×), 1M HCl (1×), H₂O (1×) and brine (1×). The organic layer wasseparated, dried over Na₂SO₄, filtered and evaporated under vacuum. Thecrude was purified by flash chromatography with 2% MeOH in DCM tofurnish 2.3 g (42%) of 9 as white solids.

¹HNMR for 9: (CDCl₃) δppm: 0.38 (1H, bs), 0.90-1.25 (m, 32H), 1.39-1.45(m, 2H), 1.50-1.60 (m, 2H), 1.70-1.82 (m, 2H), 1.96-2.01 (m, 4H),2.18-2.20 (m, 3H), 2.30-2.35 (d, 1H), 3.45-3.57 (m, 1H), 3.71-3.76 (m,1H), 4.49-4.59 (m, 1H), 4.69-4.72 (m, 1H). MS (M+H) 533.

Preparation of 10: 1.08 g (5.0 mmol) of Boc-(L)-Valine was dissolved in5 ml of DCM. To this was added 920 mg (5.0 mmols) of pentafluorophenol.The reaction was cooled in an ice-bath followed by slow addition of 0.78ml (5.0 mmols) of DIC. After complete addition the reaction mixture wasstirred at room temperature for 30 minutes. To this mixture was thenadded 532 mg (1.0 mmol) of 9 followed by 490 mg (4.0 mmol) of DMAP andthe reaction was stirred at room temperature for 24 hours. The reactionmixture was transferred into a sepratory funnel and washed with sat.NaHCO₃ (3×), 0.1N HCl (1×), H₂O (3×), and brine (1×), the organic layerwas separated, dried over Na₂SO₄, filtered and the solvent wasevaporated under vacuum. 20 ml of Et₂O was added to the residue and thewhite precipitate was filtered under suction. This operation wasrepeated once more and the filterate was evaporated and the residue waspurified using flash chromatography with solvent gradient of 2%-5% MeOHin DCM to furnish 590 mg (81%) of 10.

¹HNMR for 3: (CDCl₃) δppm: 0.35 (1H, bs), 0.53 (1H, bs), 0.75-1.30 (m,38H), 1.45 (s, 9H), 1.50-1.60 (m, 2H), 1.70-1.82 (m, 2H), 1.96-2.01 (m,4H), 2.18-2.20 (s, 3H), 2.30-2.35 (d, 1H), 3.71-3.76 (m, 1H), 4.13-4.19(m, 1H), 4.40-4.41 (m, 1H), 4.51-4.53 (m, 1H), 4.60-4.63 (m, 1H),4.69-4.72 (m, 1H), 4.81-4.83 (m, 1H). MS (M+H) 731.

Preparation of 11: 500 mg (0.68 mmols) of 10 was dissolved in 5.0 ml ofdry MeOH and 2.8 ml of 0.5 M MeONa/MeOH was added to it. The reactionwas stirred at room temperature for 24 hrs. The reaction was carefullyneutralized (monitoring with pH meter) by dropwise addition of 1MHCl/MeOH and the solvents were evaporated under vacuum. The residue wasdissolved in 30 ml of DCM and successively washed with sat. NaHCO₃ (1×),H₂O (1×), and brine (1×), dried over Na₂SO₄, filtered and the solventswere evaporated under vacuum. The crude product was purified using flashchromatography to furnish 403 mg (86%) of 11 as white solids. The crudewas carried over to the next step without any purification.

¹HNMR for 11: (CDCl₃) δppm: 0.39 (1H, bs), 0.58 (1H, bs), 0.86-1.35 (m,38H), 1.47 (s, 9H), 1.53-1.61 (m, 2H), 1.70-1.82 (m, 2H), 1.96-2.01 (m,4H), 2.30-2.35 (d, 1H), 3.19-3.22 (m, 1H), 3.71-3.76 (m, 1H), 4.13-4.19(m, 1H), 4.40-4.41 (m, 1H), 4.60-4.63 (m, 1H), 4.69-4.72 (m, 1H),4.81-4.83 (m, 1H). MS (M+H) 690.

Preparation of 12: To 400 mg (0.58 mmol) of 10 was added 0.73 ml of 4.0MHCl/dioxane and stirred for 4 hrs. The solvents were then evaporatedunder vacuum and the residue was washed with 5 mL of cold diethyl ether.The solids were filtered off and dried at high vacuum for overnight tofurnish 290 mg (80%) of 12 as white solids.

¹HNMR for 12 (DMSOd₆) δppm: 0.30 (bs, 1H), 0.45 (bs, 1H), 0.80-1.19 (m,29H), 1.47-1.58 (m, 3H), 1.61-1.70 (m, 2H), 1.81-1.89 (m, 4H), 2.19-2.30(m, 2H), 2.43-2.60 (m, 2H), 3.03-3.05 (m, 1H), 3.56-3.60 (t, 1H),3.73-3.75 (m, 1H), 3.80-4.09 (bs, 4H), 4.40-4.51 (m, 1H), 4.71-4.79 (m,1H), 8.40-8.58 (bs, 2H). MS (M+H) 590.

Example 7 Preparation of 2-(L),3-Dimethyl-pentanoic acid6α,16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester Hydrochloride salt [C3-(L)-isoleucyl-cycloastragenol]14

Preparation of 13: Boc-(L) Isoleucine-OH (Bachem, Torrance, Calif.) (1.9g. 8.16 mmols) was dissolved in 25 ml of DCM. To this was added 1.5 g(8.16 mmol) of pentafluorophenol. The reaction was cooled in an ice-bathfollowed by slow addition of 1.3 ml (8.16 mmols) of DIC. After completeaddition the reaction mixture was stirred at room temperature for 30minutes at which time the reaction mixture turned turbid(diisopropylcarbodimide-urea precipitation). To this mixture was thenadded 1.0 g (2.04 mmols) of 2 followed by 976 mg (7.0 mmol) of DMAP andthe reaction was stirred at room temperature for 24 hours. The reactionmixture was transferred into a sepratory funnel and washed with H₂O (2×)1% aq. HCl (2×), 0.1N aq. NaOH (2×), sat. NaHCO₃ (3×), H₂O (1×) andbrine (1×), the organic layer was separated, dried over Na₂SO₄, filteredand the solvent was evaporated under vacuum. The residue was purifiedusing flash chromatography with solvent gradient of 2%-5% MeOH in DCM tofurnish 943 mg (67%) of the target product 13 together with 330 mg ofthe bis product.

¹HNMR for 13: (CDCl₃) δppm: 0.38 (1H, bs), 0.52 (1H, bs), 0.93-1.28 (m,33H), 1.39-1.45 (s,m, 12H), 1.59-1.63 (m, 5H), 1.76-1.82 (m, 2H),1.96-2.01 (m, 4H), 2.16-2.20 (m, 1H), 2.30-2.35 (d, 1H), 2.49-2.54 (q,1H), 3.45-3.57 (m, 1H), 3.71-3.76 (t, 1H), 4.19-4.21 (dd, 1H), 4.53-4.61(m, 1H), 4.69-4.71 (q, 1H), 5.0-5.2 (d, 1H. MS (M+H) 704.

Preparation of 14: To a 700 mg (1.0 mmol) of the 11 was added 1.25 ml of4.0M HCl/dioxane and stirred for 4 hrs. The solvents were evaporated andthe product was precipitated in 10 ml of cold diethyl ether and thesolids were filtered. The solids were then dried under high vacuum forovernight to yield 512 mg (80%) of the target product 12 as a whitepowder.

¹HNMR for 12 (DMSOd₆) δppm: 0.36 (bs, 1H), 0.49 (bs, 1H), 0.80-1.39 (m,32H), 1.44-1.60 (m, 3H), 1.61-1.70 (m, 2H), 1.81-1.89 (m, 4H), 2.19-2.30(m, 2H), 2.41-2.60 (m, 2H), 3.29-3.41 (m, 1H), 3.58-3.61 (t, 1H),3.86-3.90 (m, 1H), 4.18-4.39 (bs, 4H), 4.51-4.53 (m, 2H), 4.54-4.59 (m,1H), 8.40-8.58 (bs, 2H). MS (M+H) 604.

Example 8 Preparation of a Mixture 2-(L)-Amino-hexanoic acid6a,16b-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3b-ylester, hydrochloride salt [C3-(L)-ornithinyl-cycloastragenol]16a;2-(L),5-Diamino-pentanoic acid3b,16b-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-6a-ylester, hydrochloride salt [C6-(L)-ornithinyl-cycloastragenol]16b and2-(L),5-Diamino-pentanoic acid3b,6a-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-16b-ylester, hydrochloride salt [C16-(L)-ornithinyl-cycloastragenol]16c

Preparation of 15a, 15b, 15c: (Boc)₂-(L)-Ornithine-OH (4.5 g, 13.6mmols) (Bachem, Torrance, Calif.) was dissolved in 15 ml of DCM. To thiswas added 2.5 g (13.6 mmol) of pentafluorophenol. The reaction wascooled in an ice-bath followed by slow addition of 2.2 ml (13.6 mmols)of DIC. After complete addition the reaction mixture was stirred at roomtemperature for 30 minutes at which time the reaction mixture turnedturbid (diisopropylcarbodimide-urea precipitation). To this mixture wasthen added 700 mg (1.43 mmols) of 2 followed by 1.6 g (13.6 mmol) ofDMAP and the reaction was stirred at room temperature for 24 hours. Thereaction mixture was transferred into a separatory funnel and washedwith H₂O (2×) 1% aq. HCl (2×), 0.1N aq. NaOH (2×), sat. NaHCO₃ (3×), H₂O(1×) and brine (1×), the organic layer was separated, dried over Na₂SO₄,filtered and the solvent was evaporated under vacuum. To the residue wasthen added 25 ml of diethylether and the urea was precipitated out. Thefiltrate was evaporated and the residue was purified using flashchromatography with solvent gradient of 2%-5% MeOH in DCM to furnish 690mg (60%) of a mixture of products 15a, 15b, 15c, and 120 mg (8%) of theC3, C6 bis product.

The ¹HNMR showed major amounts of 15a and 15b products with 2% of theregioisomer 15c.

¹HNMR of the mixture (15a, 15b and 15c): (CDCl₃) δppm: 0.38 (1H, bs),0.52 (1H, bs), 0.90-1.38 (m, 26H), 1.39-1.45 (s,m, 27H), 1.59-1.63 (m,5H), 1.76-1.82 (m, 2H), 1.96-2.01 (m, 4H), 2.16-2.20 (m, 1H), 2.30-2.35(d, 1H), 2.49-2.54 (q, 1H), 3.10-3.19 (m, 6H), 3.19-3.22 (m, 1H),3.45-3.57 (t, 1H), 3.71-3.76 (m, 1H), 4.19-4.21 (m, 1H), 4.22-4.30 (m,1H), 4.52-4.60 (m, 1H), 4.61-4.65 (m, 1H), 4.79-4.81 (m, 1H), 4.95-5.01(m, 1H), 5.13-5.21 (m, 1H), 5.38-5.41 (m, 1H). MS (M+H) 804

Preparation of 16a, 16b, 16c To a 200 mg (0.25 mmol) of the mixture of15a, 15b, 15c, was added 10 ml of 1.0M HCl/diethylether and stirred for16 hrs. The white solids were filtered and washed with 10 ml of Et₂O(4×). The solids were then dried under high vacuum for overnight toyield 160 mg (95%) of the target products 16a, 16b, 16c, as a whitepowder. The ¹HNMR showed major amounts of 16a and 16b products with 2%of the C-16 (16c) regioisomer.

The ¹HNMR of the mixture (16a, 16b and 16c): (D₂O) δppm: 0.26 (1H, bs),0.52 (1H, bs), 0.90-1.18 (m, 26H), 1.49-1.74 (m, 5H), 1.83-2.10 (m, 2H),2.20-2.31 (m, 4H), 2.81-2.92 (m, 2H), 3.19-3.20 (m, 1H), 3.39-3.42 (m,1H), 3.62-3.71 (m, 1H), 3.88-4.00 (m, 1H), 4.08-4.12 (m, 1H), 4.52-4.57(m, 1H), 4.61-4.65 (m, 1H), 4.79-4.81 (m, 1H). MS (M+H): 605.

Example 9 Preparation of a mixture of 2-(L)-Amino-pentanedioic acid1-{6a,16b-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3b-yl}ester Hydrochloride salt [C3(L0)-valyl-cycloastragenol]18a;2-(L)-Amino-pentanedioic acid1-{3b,16b-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-6a-yl}ester.Hydrochloride salt[C6-(L)-glutamate-cycloastragenol]18b2-(L)-Amino-pentanedioic acid1-{3b,6a-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-16b-yl}ester. Hydrochloride salt [C16-(L)-glutamate-cycloastragenol](18c)

Preparation of 17a, 17b, 17c: Boc-(L)-Glutamic acid (Bachem, Torrance,Calif.) (O-tBu)-OH (4.5 g, 14.82 mmols) was dissolved in 15 ml of DCM.To this was added 2.73 g (14.82 mmol) of pentafluorophenol. The reactionwas cooled in an ice-bath followed by slow addition of 2.3 ml (14.82mmols) of DIC. After complete addition the reaction mixture was stirredat room temperature for 30 minutes at which time the reaction mixtureturned turbid (diisopropylcarbodimide-urea precipitation). To thismixture was then added 765 mg (1.56 mmols) of cycloastragenol 2 followedby 1.8 g (14.82 mmol) of DMAP and the reaction was stirred at roomtemperature for 24 hours. The reaction mixture was transferred into asepratory funnel and washed with H₂O (2×) 1% aq. HCl (2×), 0.1N aq. NaOH(2×), sat. NaHCO₃ (3×), H₂O (1×) and brine (1×), the organic layer wasseparated, dried over Na₂SO₄, filtered and the solvent was evaporatedunder vacuum. To the residue was then added 50 ml of diethylether andthe urea was precipitated out. To filterate was evaporated and theresidue was purified using flash chromatography with solvent gradient of2%-5% MeOH in DCM to furnish 714 mg of (60%) of in-separable mixture ofproducts 17a, 17b, 17c, and 140 mg, (9%) of the bis product (C3, C6).The ¹HNMR showed major amounts of 17a and 17b products with 2% of theregioisomer (17c).

¹HNMR of the mixture (17a, 17b and 17c): (CDCl₃) δppm: 0.29 (1H, bs),0.56 (1H, bs), 0.90-1.20 (m, 26H), 1.30-1.40 (s,m, 25H), 1.48-1.65 (m,3H), 1.82-1.92 (m, 2H), 2.16-2.22 (m, 2H), 2.42-2.50 (m, 2H), 3.20-3.40(m, 1H), 3.55-3.61 (m, 1H), 3.82-3.96) (m, 1H), 4.42-4.50 (m, 1H),4.62-4.71 (m, 1H), 4.79-4.81 (m, 1H), 4.95-5.01 (m, 1H), 5.13-5.21 (m,1H), 5.38-5.41 (m, 1H). MS (M+H) 776.

Preparation of 18a, 18b, 18c: To a 260 mg (0.34 mmol of the mixture of17a, 17b and 17c was added 10 ml of 1.0M HCl/diethylether and stirredfor 16 hrs. The white solids were filtered and washed with 10 ml of Et₂O(4×). The solids were then dried under high vacuum for overnight toyield 210 mg (95%) of the target products 18 as a white powder. The¹HNMR showed major amounts of C-3 (18a) and C-6 (18b) products with lessthan 2% of the C-16 (18c) regioisomer.

¹HNMR of the mixture (18a, 18b and 18c): (D₂O) δppm: 0.26 (1H, bs), 0.49(1H, bs), 0.90-1.18 (m, 26H), 1.49-1.74 3.29-3.30 (m, 1H), 3.39-3.42 (m,1H), 3.62-3.71 (m, 1H), 3.88-4.02 (m, 3H), 4.52-4.57 (m, 1H), 4.61-4.65(m, 1H), 4.79-4.81 (m, 1H). MS (M+H): 620.

Example 10 Preparation of 2-(L)-Amino-3-phenyl-propionic acid6a,16b-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3a-ylester. Hydrochloride salt [C3-(L)-phenylalanyl-cycloastragenol]20a;2-(L)-Amino-3-phenyl-propionic acid3,16b-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-6a-ylester. Hydrochloride salt [C6-(L)-phenylalanyl-cycloastragenol]20b

Preparation of 19a, 19b: Boc-(L)-Phenylalanine-OH (Bachem, Torrance,Calif.) (5.0 g, 18.84 mmols) was dissolved in 30 ml of DCM. To this wasadded 3.5 g (18.84 mmol) of pentafluorophenol. The reaction was cooledin an ice-bath followed by slow addition of 2.9 ml (1.9 mmols) of DIC.After complete addition the reaction mixture was stirred at roomtemperature for 30 minutes at which time the reaction mixture turnedturbid (diisopropylcarbodimide-urea precipitation). To this mixture wasthen added 1.0 g (1.56 mmols) of cycloastragenol 2 followed by 1.8 g (15mmol) of DMAP and the reaction was stirred at room temperature for 24hours. The reaction mixture was transferred into a sepratory funnel andwashed with H₂O (2×) 1% aq. HCl (2×), 0.1N aq. NaOH (2×), sat. NaHCO₃(3×), H₂O (1×) and brine (1×), the organic layer was separated, driedover Na₂SO₄, filtered and the solvent was evaporated under vacuum. Tothe residue was then added 50 ml of diethylether and the urea wasprecipitated out. The filterate was evaporated and the residue waspurified using flash chromatography with solvent gradient of 1%-2% MeOHin DCM to furnish 950 mg of (68%) of in-separable mixture (C-3 and C-6)of products and 290 mg (30%) of the bis product (C3 and C6). The ¹HNMRshowed major amounts of C-3 (19a) and C-6 product (19b).

¹HNMR of the mixture (19a and 19b): (CDCl₃) δppm: 0.32 (1H, bs), 0.60(1H, bs), 0.70-1.20 (m, 14H), 1.30-1.40 (s,m 12H), 1.48-1.65 (m, 2H),1.76-1.92 (m, 4H), 2.22-2.32 (m, 1H), 2.52-2.58 (m, 1H), 2.86-2.91 (m,1H), 3.0-3.18 (m, 1H), , 3.20-3.24 (m, 1H), 3.55-3.61 (m, 1H), 3.72-3.80(m, 1H), 4.0-4.10 (m, 1H), 4.42-4.58 (m, 2H), 4.61-4.63 (m, 1H),4.77-4.78 (m, 1H), 4.81-4.90 (m, 1H), 7.08-7.25 (m, 5H). MS (M+H) 738.

Preparation of 20a, 20b: To a 330 mg (0.45 mmol) of the mixture of 19aand 19b was added 10 ml of 1.0M HCl/diethylether and stirred for 8 hrs.The white solids were filtered and washed with 10 ml of cold Et₂O (3×).The solids were then dried under high vacuum for overnight to yield 260mg (86%) of the target products 20a and 20b as a white powder.

¹HNMR of the mixture (20a and 20b): (DMSOd₆) δppm: 0.22 (1H, bs), 0.55(1H, bs), 0.70-1.10 (m, 24H), 1.20-1.30 (m, 3H), 1.42-1.55 (m, 2H),1.61-1.80 (m, 2H), 1.87-1.89 (m, 2H), 2.19-2.20 (d, 1H), 2.42-2.50 (m,1H), 2.92-3.10 (m, 2H), 3.20-3.21 (m, 2H), 3.30-3.34 (m, 1H), 3.55-3.61(m, 1H), 3.78-3.88 (m, 1H), 4.12-4.20 (m, 2H), 4.42-4.45 (m, 1H),4.48-4.53 (m, 1H), 4.80-4.82 (m, 1H), 7.08-7.25 (m, 5H), 8.62-8.80 (bs,3H). MS (M+H) 638

Example 11 Preparation of 3-Methyl-2-(L)-methylmino-butyric acid16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-6α-methoxy-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester, hydrochloride salt [C3-(L)-valyl-C6-cycloastragenol](22)

This analog was made starting from intermediate 3 by the followingprocedure

Preparation of 21: 280 mg (0.41 mmols) of 3 was dissolved in 1.5 mL ofNMP and 33 mg (0.82 mmols) of NaH (60% dispersion in oil) was added toit. The reaction was stirred for 10 minutes followed by addition of 80μL of dimethylsulfate and stirred at the ambient temperature for 16 hrs.The reaction mixture was diluted with 25 mL of DCM and washed with H2O(4×5 mL) and brine (1×5 mL), dried over Na2SO4 and filtered. Thefilterate was concentrated under reduced pressure. The crude waspurified purified using flash chromatography with solvent gradient of1%-3% MeOH in DCM to furnish 170 mg (58% of 21.

¹HNMR for 21: (CDCl₃) δppm: 0.38 (1H, bs), 0.52 (1H, bs), 0.90-1.38 (m,30H), 1.39-1.45 (s,m, 12H), 1.59-1.63 (m, 5H), 1.76-1.82 (m, 2H),1.96-2.01 (m, 4H), 2.16-2.20 (m, 1H), 2.30-2.35 (d, 1H), 2.49-2.54 (q,1H), 2.70-2.73 (s,s, 3H), 3.20-3.22 (s,s, 3H), 3.26-3.28 (t, 1H),3.70-3.75 (t, 1H), 4.19-4.21 (m, 1H), 4.53-4.61 (m, 1H), 4.70-4.73 (q,1H). MS (M+H) 718.

Preparation of 22: To 150 mg (0.21 mmol) of the 21 was added 8 ml of1.0M HCl/diethylether and stirred for 24 hours. The white solids werefiltered and washed with diethyl ether (2×5 ml). The solids were thendried under high vacuum for overnight to yield 115 mg (85%) of thetarget product 22 as a white powder.

¹HNMR for 22 (DMSOd₆) δppm: 0.35 (bs, 1H), 0.48 (bs, 1H), 0.81-1.40 (m,29H), 1.45-1.60 (m, 3H), 1.61-1.70 (m, 2H), 1.81-1.89 (m, 4H), 2.19-2.30(m, 2H), 2.41-2.70 (m, 5H), 3.0-3.18 (m, 3H), 3.20-3.28 (m, 2H),3.58-3.61 (t, 1H), 3.81-3.83 (m, 1H), 4.0-4.12 (bs, 4H), 4.49-4.51 (m,1H), 4.62-4.70 (m, 1H), 9.20-9.42 (bs, 2H). MS (M+H) 618.

Example 12 Preparation of 2-(L)-Amino-3-methyl butyric acid6α,16β-dimethoxy-17-[5-(1-methoxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester. Hydrochloride salt[C3-(L)-valyl-C6,C16-dimethoxy-cycloastragenol]27

Preparation of 23: 5.0 g (10.2 mmols) of 2 was dissolved in pyridine (50ml) and cooled to 0° C. Benzoyl chloride (2.35 ml, 20.4 mmols) was addedand the reaction mixture was stirred at room temperature for 24 hrs. Thereaction mixture was diluted with 200 ml of diethyl ether and washedwith sat. NaHCO₃ (2×), H₂O (2×) and brine (1×). Dried over MgSO₄,filtered and the solvents evaporated under vacuum. The crude waspurified by column chromatography using 1%-2% MeOH in DCM to furnish 1.6g (26%) of 23 as white solids.

¹HNMR for 23: (CDCl₃) δppm: 0.38 (bs, 1H), 0.55 (bs, 1H), 0.90-2.0 (m,37H), 1.70-1.82 (m, 2H), 2.30-2.35 (m, 1H), 2.50-2.6 (m, 1H), 3.45-3.57(m, 1H), 3.71-3.76 (m, 1H), 4.69-4.71 (m, 1H), 4.79-4.81 (m, 1H), 7.41(t, 2H), 7.52 (t, 1H), 8.03 (d, 2H). MS (M+H) 595.

Preparation of 24: 600 mg (1.01 mmols) of 23 was dissolved in THF (10ml) and NaH (323 mg, 8.08 mmols) was added and the reaction mixture wasstirred for 20 minutes. Dimethyl sulfate (509 mg, 4.04 mmols) was addedand the reaction mixture was stirred at room temperature for 16 hrs. Thereaction mixture was diluted with 100 ml of diethyl ether and quenchedwith H₂O and successively washed with H₂O (2×) and brine (1×), driedover MgSO₄, filtered and the solvents evaporated under vacuum. The crudewas purified by column chromatography using 1%-2% MeOH in DCM to furnish460 mg of (72%) of 24 as white solids.

¹HNMR for 24. (CDCl₃) δppm: 0.30 (bs, 1H), 0.53 (bs, 1H), 0.90-2.0 (m,37H), 2.40-2.45 (m, 2H), 2.92-2.96 (m, 1H), 3.10-3.14 (s, 3H), 3.22-3.24(s, 3H), 3.80-3.82 (m, 1H), 3.9-4.10 (m, 1H), 4.69-4.70 (m, 1H), 7.43(t, 2H), 7.52 (t, 1H), 8.04 (d, 2H). MS (M+H) 637.

Preparation of 25: 460 mg (0.72 mmols) of 24 was dissolved in DCM (10ml) and to this was added 10 ml of 0.5M solution of NaOMe in MeOH andthe reaction mixture was stirred at 40° C. for 48 hrs. The reactionmixture was quenched with a solution of sat. NaHCO₃ and the solventswere then evaporated under reduced pressure. The crude was purified bycolumn chromatography using 1%-2% MeOH in DCM to furnish 210 mg of (55%)of 25 as white solids.

¹HNMR for 25: (CDCl₃) δppm: 0.24 (bs, 1H), 0.49 (bs, 1H), 0.90-2.0 (m,37H), 2.40-2.45 (m, 2H), 2.92-2.96 (m, 1H), 3.10-3.14 (m, 3H), 3.22-3.24(m, 3H), 3.26-3.28 (m, 1H), 3.80-3.82 (m, 1H), 3.9-4.10 (m, 1H). MS(M+H) 533.

Preparation of 26: Boc-Val-OH (685 mg, 3.16 mmols) was dissolved in 3 mlof DCM. To this was added 581 mg (3.16 mmol) of pentafluorophenol. Thereaction was cooled in an ice-bath followed by slow addition of 0.49 ml(3.16 mmols) of DIC. After complete addition the reaction mixture wasstirred at room temperature for 10 minutes at which time the reactionmixture turned turbid (diisopropylcarbodimide-urea precipitation). Tothis mixture was added 210 mg (0.395 mmols) of 25 followed by 385 mg(3.16 mmol) of DMAP and the reaction was stirred at room temperature for48 hours. The reaction mixture was transferred into a separatory funneland washed with H₂O (2×) 1% aq. HCl (2×), 0.1N aq. NaOH (2×), sat.NaHCO₃ (3×), H₂O (1×) and brine (1×), the organic layer was separated,dried over Na₂SO₄, filtered and the solvent was evaporated under vacuum.To the residue was then added 10 ml of diethylether and the urea wasprecipitated out. The filtrate was evaporated and the residue waspurified using flash chromatography with solvent gradient of 2%-5% MeOHin DCM to furnish 277 mg (96%) of the target product 26.

¹HNMR for 26: (CDCl₃) δppm: 0.23 (bs, 1H), 0.49 (bs, 1H), 0.90-2.0 (m,52H), 2.20-2.25 (m, 1H), 2.32-2.45 (m, 1H), 2.92-3.0 (m, 1H), 3.08-3.10(s, 3H), 3.19-3.20 (s, 3H), 3.22-3.25 (s, 3H), 3.82-3.84 (m, 1H),3.90-3.92 (m, 1H), 4.10-4.21 (m, 1H), 4.50-4.58 (m, 1H), 4.91-5.01 (m,1H). MS (M+H) 732.

Preparation of 27 To 100 mg (0.14 mmol) of the 26 was added 8 ml of 1.0MHCl/diethylether and stirred for 8 hrs. The white solids were filteredand washed with diethyl ether (2×5 ml). The solids were then dried underhigh vacuum for overnight to yield 65 mg (70%) of the target product 27as a white powder.

¹HNMR for 27 (DMSOd₆) δppm: 0.20 (bs, 1H), 0.38 (bs, 1H), 0.75-1.90 (m,43H), 2.10-2.15 (m, 1H), 2.20-2.25 (m, 1H), 2.82-2.88 (m, 1H), 2.93-3.03(m, 3H), 3.19-3.20 (s, 3H), 3.22-3.25 (s, 3H), 3.70-3.79 (m, 1H),3.90-3.92 (m, 1H), 4.10-4.21 (m, 1H), 4.50-4.58 (m, 1H), 8.14-8.24 (bs,3H). MS (M+H) 632.

Example 13 Preparation of 2-(L)-Amino-3-methyl butyric acid16β-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-3-oxo-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-6α-ylester, hydrochloride salt [C6-(L)-valyl-cycloastragenol](30):Preparation of C3-(L)-valyl-cycloastragenone

Preparation of 28: To a stirred solution of DMSO (6.4 g, 4 equ, 100 mLof DCM) at −60 to −70° C., oxalyl chloride (5.2 g in 10 mL of DCM) wasadded and it was stirred for 10 minutes. Compound 2 (10 g in 200 mL ofDCM) was added over a period of 10 minutes and the reaction mixture wasstirred for 30 minutes followed by addition of triethylamine (10.3 gover 5 min). The reaction was stirred at −60 to −70° C. for 1-2 hrsuntil the reaction was complete. The crude product 28 was purified bycolumn chromatography. Eluted with Petroleum ether:ethyl acetate=4:1 toget 8 g of mono-oxidation product.

¹HNMR for 28: (CDCl₃) δppm: 0.38 (bs, 1H), 0.58 (bs, 1H), 0.80-1.32 (m,25H), 1.50-2.20 (m, 12H), 2.30-2.70 (m, 4H), 2.50-2.6 (m, 1H), 3.45-3.52(m, 1H), 3.71-3.76 (m, 1H), 4.69-4.72 (m, 1H), MS (M+H) 489.

Preparation of 29: Boc-(L) Val-OH (0.54 g) was dissolved in 15 ml ofDCM. To this solution 0.45 g of pentafluorophenol was added. Thereaction was cooled in an ice-bath followed by slow addition of 0.4 mlof DIC. After complete addition the reaction mixture was stirred at roomtemperature for 30 minutes at which time the reaction mixture turnedturbid (diisopropylcarbodimide-urea precipitation). To this mixture wasthen added 0.3 g of compound 28 followed by 0.3 g of DMAP and thereaction was stirred at room temperature for 24 hours. The reactionmixture was transferred into a separatory funnel and washed with 0.1Naq. NaOH (2×), H₂O (3×) and brine (1×), the organic layer was separated,dried over Na₂SO₄, filtered and the solvent was evaporated under vacuum.The residue including compound 29 was not purified but taken to the nextstep of deprotection.

Preparation of 30: The above crude product was treated with HCl in ethylacetate for 12 hrs. The product was then isolated by extraction withwater and upon drying provided the crude HCl salt which was purified byprep-HPLC with petroleum ether and ethyl acetate mixture. The purefractions were pooled to provide 120 mg of the final product 30.

¹HNMR for 30. (DMSOd₆) δppm: 0.40 (bs, 1H), 0.80 (bs, 1H), 0.80-1.32 (m,29H), 1.50-2.20 (m, 12H), 2.30-2.32 (m, 2H), 2.40-2.45 (m, 2H),3.60-3.62 (m, 1H), 3.93-4.01 (m, 1H), 4.49-4.51 (m, 1H), 4.79-4.81 (m,1H). MS (M+H) 588.

Example 14 Preparation of 2-(L)-Amino-3-methyl-pentanoic acid6α-(2-amino-3-methyl-pentanoyloxy-16β-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester hydrochloride salt [C3,C6-(L,L)-bis-isoleucine-cycloastragenol]-32

Preparation of 31: Boc-(L)-Ile-OH (4.6 g, 20 mmols) was dissolved in 25ml of N-methylpyrrolidone (NMP). To this was added 3.7 g (20 mmol) ofpentafluorophenol. The reaction was cooled in an ice-bath followed byslow addition of 3.1 ml (20 mmols) of DIC. After complete addition inthe reaction mixture was stirred at room temperature for 30 minutes atwhich time the reaction mixture turned turbid(diisopropylcarbodimide-urea precipitation). To this mixture was thenadded 1.0 g (2.04 mmols) of 2 followed by 1.7 g (14 mmol) of DMAP andthe reaction was stirred at room temperature for 24 hours. The reactionmixture was transferred into a sepratory funnel and washed successivelywith H₂O (6×), 1% aq. HCl (2×), 0.1N aq. NaOH (2×), sat. NaHCO₃ (3×),H₂O (1×) and brine (1×), the organic layer was separated, dried overNa₂SO₄, filtered and the solvent was evaporated under vacuum. Theresidue was purified using flash chromatography with solvent gradient of2%-5% MeOH in DCM to furnish 1.4 g (80%) of the target product 31.

¹HNMR for 31: (CDCl₃) δppm: 0.38 (1H, bs), 0.60 (1H, bs), 0.80-1.0 (m,24H), 1.13 (s,s, 6H), 1.20 (s,s, 6H), 1.32 (s, 6H), 1.35 (s,s, 4H), 1.41(s,s, 18H), 1.55-1.60 (m, 6H), 1.79-1.83 (m, 3H), 3.71-3.75 (t, 1H),4.08-4.20 (m, 2H), 4.58-4.60 (m, 1H), 4.61-4.71 (q, 1H), 4.72-4.80 (m,1H), 4.82-4.84 (d, 1H), 4.9-5.0 (d, 1H). MS (M+H) 915.

Preparation of 32: To a 1.5 g (1.6 mmol) of the 31 in 4 ml of dry Et₂Owas added 3.5 ml of 4.0M HCl/dioxane and stirred for 4 hrs. The solventswere evaporated and the product was precipitated with three times of 40ml of cold diethyl ether and the solids filtered off. The solids werethen dried under high vacuum for overnight to yield 3.1 g (91%) of thetarget product 32 as a white powder.

¹HNMR for 32 (DMSOd₆) δppm: 0.22 (bs, 1H), 0.57 (bs, 1H), 0.80-1.20 (m,35H), 1.41-1.80 (m, 14H), 2.10-2.21 (m, 2H), 2.34-2.42 (m, 4H),2.20-2.30 (m, 2H), 3.59-3.62 (m, 1H), 3.81-3.83 (m, 2H), 4.42-4.53 (m,1H), 4.61-4.71 (m, 1H), 4.81-4.9 (m, 1H), 8.40-8.70 (d, 4H). MS (M+H)717.

Example 15 Preparation of 2-(L)-Amino-3-methyl-butyric acid3β,6α-dihydroxy-16β-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-16β-ylester hydrochloride salt [C16-(L)-Valyl-cycloastragenol]-36

Preparation of 33: To 4 g (8.2 mmol) of 2 was added 100 ml of pyridineand cooled in an ice-bath. To this was slowly added 77 ml (820 mmol) ofacetic anhydride followed by 100 mg (0.81 mol) of DMAP. The reactionmixture was stirred at for 16 hrs. The reaction mixture was cooled in anice bath and quenched with 3% aq. HCl and diluted with 200 ml of DCM andwashed successively with the following: sat. aq. NaHCO₃, (2×), H₂O (3×)and brine (1×). The organic layer was separated, dried over Na₂SO₄,filtered and evaporated under vacuum. The crude was purified by flashchromatography with 1% MeOH in DCM to furnish 4.2 g (89%) of 33 as whitesolids.

¹HNMR for 33: (CDCl₃) δppm: 0.38 (1H, bs), 0.49 (1H, bs), 0.90-1.25 (m,32H), 1.39-1.45 (m, 2H), 1.50-1.60 (m, 2H), 1.70-1.82 (m, 2H), 1.96-2.01(m, 4H), 2.18-2.20 (s,s, 6H), 2.30-2.35 (d, 1H), 3.71-3.76 (m, 1H),4.49-4.59 (m, 1H), 4.69-4.72 (m, 2H). MS (M+H) 575.

Preparation of 34: 1.54 g (2.7 mmol) of 33 was dissolved in 8.0 ml ofNMP. To the clear solution was added 800 mg (8.3 mmol) of potassiumtert-butoxide and stirred for 45 mins. To this was added 3.0 g (8.9mmols) of Boc-Val-ONp followed by 245 mg (2 mmol) of DMAP and stirredfor 24 hrs. The reaction mixture was diluted with 100 ml of DCM andwashed successively with the following: H₂O (4×), 1% aq. HCl (1×), (sat.aq. NaHCO₃, (2×), H₂O (3×) and brine (1×). The organic layer wasseparated, dried over Na₂SO₄, filtered and evaporated under vacuum. Thecrude was purified by flash chromatography with petroleum ether/ethylacetate to furnish 1.0 g (48% of 34 as white solids.

¹HNMR for 34: (CDCl₃) δppm: 0.38 (1H, bs), 0.49 (1H, bs), 0.90-1.25 (m,38H), 1.39-1.45 (m,s, 11H), 1.50-1.60 (m, 2H), 1.70-1.82 (m, 2H),1.96-2.01 (m, 4H), 2.18-2.20 (s,s, 6H), 2.30-2.35 (d, 1H), 3.71-3.76 (m,1H), 4.13-4.18 (m, 1H), 4.49-4.59 (m, 1H), 4.62-4.72 (m, 1H), 5.12-5.17(m, 1H), 5.38-5.42 (m, 1H). MS (M+Na⁺) 796

Preparation of 35: To 700 mg (0.91 mmol) of 34 was added 20 ml of 0.5Msolution of MeOH/MeONa and stirred for 16 hrs. The reaction was cooledin an ice bath and quenched with a solution of 1% aq. HCl to a pH of 5.The methanol was evaporated under reduced pressure and to the aq. layerwas added a solution of saturated aq. NaHCO₃ and extracted with DCM(4×), the combined organic layer was dried over Na₂SO₄, filtered andevaporated under vacuum. The crude was purified by flash chromatographywith a gradient of 2% to 3% MeOH/DCM to furnish 360 mg (58%) of 35 aswhite solids.

¹HNMR for 35: (CDCl₃) δppm: 0.38 (1H, bs), 0.49 (1H, bs), 0.90-1.25 (m,38H), 1.39-1.45 (m,s, 11H), 1.50-1.60 (m, 2H), 1.70-1.82 (m, 2H),2.10-2.20 (m, 2H), 2.30-2.35 (d, 1H), 3.20-3.25 (m, 1H), 3.41-3.50 (m,1H), 3.71-3.76 (m, 1H), 4.13-4.18 (m, 1H), 5.12-5.17 (m, 1H), 5.38-5.42(m, 1H). MS (M+Na⁺) 712

Preparation of 36: To 350 mg (0.51 mmol) of 35 was added 10 ml of 1.0 Msolution of HCl/Et₂O and stirred for 5 hrs. The solvents were evaporatedunder reduced pressure and the residue was washed with dry 10 ml of Et2O(3×) and filtered under vacuum. The white solids were dried under highvacuum to furnish 25 mg (78%) of 36 as white solids.

¹HNMR for 36: (DMSOd₆) δppm: 0.38 (1H, bs), 0.49 (1H, bs), 0.80-1.25 (m,29H), 1.39-1.83 (m, 4H), 2.10-2.20 (m, 2H), 2.30-2.40 (m, 4H), 3.18-3.21(m, 1H), 3.38-3.40 (m, 1H), 3.71-3.76 (m, 1H), 4.13-4.17 (m, 1H),5.40-5.42 (m, 1H), 8.38-8.53 (bs, 3H). MS (M+H) 590

Biological Example 1 Keratinocyte Cell/Telomerase Repeat AmplificationProtocol (TRAP) Assay

The ability of a compound to increase telomerase activity in a cell canbe determined using the TRAP (Telomeric Repeat Amplification Protocol)assay, which is known in the art (e.g. Kim et al., U.S. Pat. No.5,629,154; Harley et al., U.S. Pat. No. 5,891,639). The activity istypically compared to the activity similarly measured in a control assayof such cells (e.g., a telomerase activity 50% greater than observed ina solvent control).

Cell lines suitable for use in the assay, normal human fibroblasts (NHF)or normal human keratinocytes (NHK), can be obtained from commercialsources, such as Cascade Biologics, Portland, Oreg. or 4C Biotech,Seneffe, Belgium, or from the ATCC (American Type Culture Collection).ATCC normal human fibroblast cell lines, which can be located on theATCC web site, include, for example, CCL135, CCL137, and CCL 151.

Human epidermal keratinocyte (neonatal HEK) from three individual donors(Cascade Biologics, Portland, Oreg.) were pooled together and a WorkCell Bank generated. the cells were cultured in EpiLife Medium (CascadeBiologics, Cat.#M-EPI-500, Portland, Oreg.) supplemented with HKGS(Human Keratinocyte Growth Supplement) (Cascade, Cat.#S-001-5). HEKneo-Pcells were seeded in 96-well plate 24 hr before treatment bytrypsinizing the cells and neutralizing the digestion by neutralizationbuffer TN® (Cascade Biologics, Portland, Oreg.) to make a cellsuspension. The cells were seeded at 5000 cells/100 uL/well in growthmedium and the plate incubated at a 37° C., 5% CO₂/95% air, in ahumidified tissue culture incubator. When the cells reach 75-80%confluence, seeding density should be around 2.5×10³/cm².

Compounds to be tested were formulated in 10% DMSO with desiredconcentrations. 11 μL of the formulated compound in a concentration of0.01 to 10 μM was added to the 96-well culture along with a control of11 μL 10% DMSO. Non-treatment control (NT) was also included. Cells wereharvested at 24 hr+/−1 hr by removing the growth medium and washing oncewith PBS (phosphate buffered saline) removing as much medium aspossible. The cells were lysed by adding 50 μL of M-Per buffer (PierceCat# 78503 & 78501) and incubating on ice for 1 hr+/−15 min. The platewas, optionally, centrifuged at 2000 RPM, 5 min. The lysate wascarefully collected from each well of the plate and transferred to afresh V-bottom storage 96-well plate, leaving the monolayer cellsintact.

A cytotoxicity assay was performed in parallel with the cell lysis bypreparing a duplicate cell culture plate treated with the samecompounds. After 24 hours+/−1 hour of incubation with compounds, 11 μL1× Alamar Blue was added to the duplicate plate and the plate wasincubated at 37° C. The plate was read at 1 and 3 hr with a fluorescenceplate reader with excitation wavelength at 530 nm and emissionwavelength at 590 nm. Cell viability (cytotoxicity) was directlyproportional to the Alomar Blue reading.

Tris-HCl pH 8.3200 mM MgCl2 15 mM KCl 650 mM Tween 20 0.5% EGTA 10 mMBSA 1 mg/ml

Primers:

Cy5-TS primer (AAT CCG TCG AGC AGA GTT) 5′ end labeled (SEQ ID NO:1)

ACX primer (GCGCGCCTTACCCTTACCCTTACCCTAACC) (SEQ ID NO:2)

Taq polymerase was AmpliTaq DNA Polymerase, (Applied Biosystems, cat. #N8080171) and dNTP (Invitrogen, cat. # R72501).

TABLE 1 TRAP assay set up Stock concentration Per Reaction (μL) Finalcon. 10x TRAP buffer w/ 5 1x BSA dNTP 2.5 mM 1   50 uM Cy5-TS Primer 0.5mg/ml, 83 μM 0.1   1 ng/μL ACX 0.1 mg/ml, 11 μM 1   2 ng/μL Taqpolymerase   5 U/ul 0.4 0.04 U/μL cell extract  5-10 H₂O 32.5-37.5 Total45

The PCR mix includes the following components: Cy5-TS primer, a 5′-Cy5labeled oligonucleotide have the sequence 5′-AAT CCG TCG AGC AGA GTT-3′(SEQ ID NO:1), is a telomerase substrate. Depending on the telomeraseactivity of the medium, telomer repeats (having the sequence . . .AGGGTT . . . ) will be added to the substrate, to form telomeraseextended products, also referred to as telomerase products or TRAPproducts. The ACS primer, having the sequence 5′-GCG CGG CTT ACC CTT ACCCTT ACC CTA ACC-3′ (SEQ ID NO:2), is an anchored return primer thathybridizes to the telomerase extended products.

A sample of cell lysate (5-10 μL) was added to the PCR mix in a reactiontube, and telomere extension/amplification is carried out by incubatingthe mixture at the following temperatures for the times indicated 30° C.30 min; then 28 cycles of the 3-step PCR reaction; 94° C. for 30seconds, 60° C. for 30 seconds, 72° C. for 1 minute, followed by 72° C.for 4 minutes, and hold at 4° C. The PCR reaction products are ready tosubject to run polyacrylamide gel electrophoresis.

Loading dye containing e.g. bromophenol blue and xylene cyanol was addedto the reaction mixture, and the samples are subjected to 10-15%non-denaturing Polyacrylamide gel electrophoresis (PAGE) in 1×TBE. TheTRAP reaction products are observed, e.g. by using a fluoroimager fordetection of CY5-labeled telomerase products (maximal excitation at 650nm; maximal emission at 670 nm)

Telomerase activity was measured by captured total pixel vol. (DNAladder bands) above background for each gel lane. The activity wasnormalized by measuring the total RNA (ng/mL) by using Ribogreen® RNAQuantitation Kit from Molecular Probes, cat. # R-11490 and followingcommercially recommended conditions with an RNA standard range of0.8-2300 ng/mL, 1:2000 dilution of RG dye, 100-250× dilution of sample.

Total Pixel Vol/RNA=Normalized Relative Telomerase Activity

Cells viability (cytotoxicity) was directly proportional to the ABreading.

The results are shown in table 2.

TABLE 2 Activity in in EC₅₀ and Com- vitro fold pound HEK increase of #Name Structure assay activity  2 cycloastragenol

+ EC₅₀ 30 nM Max: 3.3 fold  4 C3-(L)-Valyl- cycloastragenol MW = 624.5

+ EC₅₀  6-22 nM Max 4.5 fold  7 C3,C6-(L,L)- bisvalyl- cycloastragenolMW = 761   

+ EC₅₀ 41-50 nM; Max. 4.8 fold 12 C6-(L)-Valyl- cycloastragenol MW =624.5

+ EC₅₀ 28-32 nM: Max. 4.1 fold 14 C3-(L)- Isoleucyl- cycloastragenol MW= 639   

+ EC₅₀  9-21 nM; Max. 4.0 fold 18a, 18b, 18c C3-(L)- Glutamate-cycloastragenol, C6-(L)- Glutamate- cycloastragenol, L-Glutamate- C16-cycloastragenol MW = 654.5

Active in PBMC

16a, 16b, 16c C3-(L)- Ornithinyl- cycloastragenol, C6-(L)- Ornithinyl-cycloastragenol, C16-(L)- Ornithinyl- cycloastragenol MW = 677   

−

20a, 20b C3-(L)- phenylalanyl- cycloastragenol, C6-(L)- phenylalanyl-cycloastragenol MW = 637.5

Active in PBMC

32 C3,6 (L)- isoleucyl- cycloastragenol

+ Max 3    fold at 0.37-1.1  μM 36 C16-(L)-valyl- cycloastragenol MW =624.5

+ Max. 3.0 fold at 0.01-0.12 μM 30 C6-(L)-valyl- C3-cyclo astragenone

+ EC₅₀ 31 μM Max 3.5 22 C3-(L)-N- Methyl Valyl- C6 Methoxy-cycloastragenol MW = 652.5

−  8 C3,C6-(D,D)- bisvalyl- cycloastragenol MW = 761   

−  5 C3-(D)-valyl- cycloastragenol MW = 624.5

− C16-(L)-valyl- C3,6-diacyl- cycloastragenol MW = 710.5

− C6-(L)-valyl- C3-acyl- cycloastragenol MW = 667.5

− 27 C3-(L)-valyl- C6,16- dimethoxy- cycloastragenol MW = 652.5

− + telomerase activation is 2 fold or more in comparison with vehiclecontrol at the peak of the full dose curve.

Biological Example 2 Peripheral Blood Monocyte Cell/Telomerase RepeatAmplification Protocol (TRAP) Assay

PBMC Isolation. Blood was collected in sodium heparin vacutainers andpooled into a single 50 mL polypropylene tube. Blood was diluted 1:1with 1×PBS and mixed thoroughly by inversion. 25 mL of diluted blood waslayered over 12 mL of Lympholyte-H (Cedarlane Laboratories) andcentrifuged at room temperature for 20 min at 800 g. Using a pipette,lymphocyte layer at interface of Lympholyte-H was carefully removed andtransferred to a new 50 mL tube. The transferred cells were diluted 1:1with 1×PBS and centrifuged at 800 g for 10 min to pellet thelymphocytes. The lymphocytes were washed 2 times with “complete” media,which consists of RPMI (Sigma, cat. No. R8758) that has beensupplemented with 10% heat-inactivated FBS and 10 mM Hepes.

Culture Conditions. The cells were counted using Trypan Blue exclusionand resuspended in complete media that is supplemented with 50 Units ofhIL-2/mL so that final concentration of cells is 1×10⁶/mL. To cellsuspension, CD2/3/28 Ab-coated beads from the T cellactivation/expansion kit (Miltenyi, cat. No. 130-091-441) was added at aratio of 1:2 (bead:cell). Cells were grown in a flask and half of themedia is changed every 2-3 days (along with 20 Units of hIL-2/mL). Atleast once a week, cells are counted and media level is adjusted to keepcells around 5—10⁵/mL.

Formulation of Analogs. Analogs were formulated in pure culture-gradeDMSO at a concentration of 1 mM. From this stock, analogs are diluted to100 μM in complete RPMI medium. A portion of the 100 uM formulation wasdiluted to 10 μM in complete RPMI medium containing 10% DMSO. Also,vehicle control was formulated by diluting DMSO in complete RPMI mediato obtain a 10% solution (this is equivalent to the amount of DMSO inthe analog dilutions).

Treatment with Analogs. After 10-14 days in culture, cells were countedand resuspended in conditioned media at a concentration of 1×10⁶/mL. 0.5mL of this cell suspension was plated into wells of a 24-well plate.Analog was diluted in fresh complete RPMI media 1:50 as to obtainconcentrations of 2 μM (from 100 μM stock) and 0.2 μM (from 10 μMstock). Also, dilute the vehicle control (10% DMSO in RPMI medium) 1:50in fresh complete RPMI media. Each well that contains 0.5 mL of cellsuspension (should be 5×10⁵ per well) receives 0.5 mL of diluted analogor DMSO vehicle control. Final concentrations of analogs were therefore1 μM and 0.1 μM and final concentration of DMSO in all wells (includingthe vehicle control) is 0.1%.

Cell Harvesting and Preparation of cell Lysate. 24 hours after additionof analogs and DMSO vehicle control to culture, cells were removed fromwells and added to microfuge tubes. Cells were centrifuged at 14,000 rpmfor 2 minutes and media was aspirated, followed by resuspension in 0.5mL of cold 1×PBS. Cells were centrifuged again for 2 minutes and PBS wasaspirated. Cell pellet was resuspended in 100 μL of M-PER (mammalianprotein extraction reagent) and incubated on ice for 30 minutes. Afterincubation, suspension was centrifuged at 14,000 rpm for 20 mm at 4° C.Following spin, 80 μL of lysate was transferred to a pre-chilledmicrofuge tube, being careful not to transfer any cellular debris. Finalconcentration of cell lysate was 5000 cells/μL.

TRAP Reaction and Gel. Samples were analyzed using a 1-step TRAP PCRreaction. Before conducting the reaction, samples were diluted 1:5 inM-PER buffer (1000 cells/μL). For each reaction the following mixturewas used: 37.5 μL of H₂O, 5 μL of 10×TRAP buffer with BSA, 1 μL of 2.5mM dNTP, 1 μL of 0.1 mg/mL ACX primer, 0.1 μL of 0.5 mg/mL Cy5-labeledTS primer, 0.4 μL of 5 U/μL Taq Polymerase, and 5 μL of diluted sample(50 μL total reaction). PCR reaction was as follows: 30° C. for 30minutes, 28 cycles of 94° C. for 30 seconds, 60° C. for 30 seconds, 72°C. for 1 minutes, followed by 72° C. for 4 minutes. PCR products wereseparated on a 12.5% polyacrylamide gel and analyzed using a STORMphosphorimager.

Activity in Fold increase Compound Name PBMC at 1 μM 2 cycloastragenol++  1.6-20.7 4 C3-(L)-Valyl- ++  1.4-19.4 cycloastragenol MW = 624.516a, 16b, 16c C3-(L)-Ornithinyl- + 1.2-8.1 mixture cycloastragenol,C6-(L)-Ornithinyl- cycloastragenol, C16-(L)-Ornithinyl- cycloastragenolMW = 677 18a, 18b, 18c C3-(L)-Glutamate- + 0.9-6.8 mixturecycloastragenol, C6-(L)-Glutamate- cycloastragenol, L-Glutamate-C16-cycloastragenol MW = 654.5 20a, 20b C3-(L)-phenylalanyl- −/+ 2.0-4.1mixture cycloastragenol, C6-(L)-phenylalanyl- cycloastragenol MW = 637.5

Biological Example 3 Administration of Compounds to Mice and Analysis ofPlasma Levels and Telomerase Activity in Tissues

The plasma levels of a compound following a single intravenous, oral,intra-peritoneal, or sub-cutaneous administration in male C57BL/6 micewas determined. Plasma samples were collected and used to determine theplasma concentration of the compound and metabolites. In addition tissuesamples, including whisker samples, and peripheral blood mononuclearcells (PBMC) cells were collected for telomerase activity analysis.

C57BL16 mice were divided into treatment groups. The mice were providedad libitum SLAC-MO1 #W080208 (Shanghai Laboratories Animal Center,Shanghai, China) throughout the in-life portion of the study with theexception of the overnight fasting period prior to oral dosing. Waterwas available ad libitum.

Environmental controls for the animal room were set to maintain atemperature of 23±2° C., humidity of 50-70%, and a 12-hour light/12-hourdark cycle. The 12-hour dark cycle may be temporarily interrupted toaccommodate study procedures. Animals were acclimated to studyprocedures for 1-7 days prior to initial dose administration.

Animals used in this study were selected based on body weights that fallwithin ±20% of the mean body weight, overall health and acclimation tocaging. Animals were given free access to both food and water during thewhole course of study with the exception of the overnight fasting periodprior to oral dosing.

Doses were administered intravenously via tail vein, orally,sub-cutaneous, or intra-peritoneally as indicated in Table 3. Bodyweights were taken on the day of dose administration. Dose volume wasdetermined based on individual body weight taken on day of dosing.

Blood samples (approximately 300 μL) were collected via cardiac punctureor via retro-orbital puncture after anesthesia into tubes containingK2-EDTA anticoagulant and 1 mg/ml NaF at the various time points afterdosing. Blood was stored on ice and then plasma separated viacentrifugation (8000 rpm×6 minutes). The plasma was stored at 20° C.until LC-MS/MS analysis.

Euthanasia was done by carbon dioxide inhalation followed byexsanguination. Whisker and peripheral blood mononuclear cells (PBMC)were collected in some animals at 30 hrs and were stored at −80° C.after processing.

PBMCs were harvested from blood using K₂EDTA as the anticoagulant. Aftercollection, the tube was gently inverted 8-10 times to mix. The tube wascentrifuged at 12000 rpm for 30 sec. to pellet cells, the supernatantwas removed and the resulting PBMC pellets were flash frozen in dryice/methanol and were stored at −80° C. Cells were processed asindicated in Biological Example 2. FIG. 1 shows the telomerase activityin PBMCs over time after treatment with compound 4.

A single or group of whiskers were plucked and 10-20 whiskers/animalwere placed in 200 μL of M-Per buffer (Pierce catalog#:78503/78501/78505, submerging the follicles). The samples were frozenin dry ice/methanol within 1 hour of plucking. FIG. 2 shows thetelomerase activity in whiskers over time after treatment with compound4.

It was determined that the mono-amino acid substituted compounds ofFormula I when administered to mice show some conversion tocycloastragenol and the di-amino acid substituted compounds may show aminor amount of conversion to monosubstituted compounds.

The positional isomer mixtures, C3-(L)-Ornithinyl-cycloastragenol,C6-(L)-Ornithinyl-cycloastragenol, C16-(L)-Ornithinyl-cycloastragenol,(16a, 16b, 16c mixture) and C3-(L)-Glutamate-cycloastragenol,C6-(L)-Ornithinyl-cycloastragenol, L-Glutamate-G16-cycloastragenol (18a,18b, 18c mixture) were not bioavailable in mice.

The bioavailability of the compounds is shown in Table 3.

TABLE 3 Study and bioavailability in Mice Dose Test Level DosingBioavailability compound (mg/kg) Vehicle route % F 4 10 2% EtOH/98%water PO 48 4 10 2% EtOH/98% water SC 66 7 10 2% EtOH/98% water PO 25 710 2% EtOH/98% water SC 61 12 5 2% EtOH/98% water PO 8 12 5 2% EtOH/98%water IP 42 14 10 5% PEG400/5% solutol PO 42 HS-15/90% water

Biological Example 4 Administration of Compounds to Male Rats andAnalysis of Plasma Levels and Telomerase Activity in Tissues

The plasma levels of a compound following a single intravenous and oraladministration in carotid artery-cannulated male Sprague Dawley rats wasdetermined. Plasma samples were collected and used to determine theplasma concentration of the compound and metabolites. In addition tissuesamples, including whisker samples and PBMC cells were collected fortelomerase activity analysis.

Carotid artery-cannulated male Sprague Dawley rats were divided intotreatment groups according to Table 6. The rats were provided ad libitumSLAC-MO1 # YY080208 (Shanghai Laboratories Animal Center, Shanghai,China) throughout the in-life portion of the study with the exception ofthe overnight fasting period prior to oral dosing. Water was availablead libitum.

Environmental controls for the animal room were set to maintain atemperature of 23±2° C., humidity of 50-70%, and a 12-hour light/12-hourdark cycle. The 12-hour dark cycle may be temporarily interrupted toaccommodate study procedures. Animals were acclimated to studyprocedures for 1-7 days prior to initial dose administration.

Animals used in this study were selected based on body weights that fallwithin ±20% of the mean body weight, overall health and acclimation tocaging. Animals were given free access to both food and water during thewhole course of study with the exception of the overnight fasting periodprior to oral dosing.

The compounds were dissolved in 2% EtOH/98% water to yield a finalconcentration of 2.5 mg/ml and 1 mg/ml for both intravenous and oraladministration, respectively. The concentration of each compound wasconfirmed by HPLC analysis.

Doses were administered intravenously via tail vein and orally asindicated in Table 4. Body weights were taken on the day of doseadministration. Dose volume was determined based on individual bodyweight taken on day of dosing.

Blood samples (approximately 250 μL) were collected via cannulae intotubes containing K2-EDTA anticoagulant and 1 mg/ml NaF at the varioustime points. Blood was stored on ice and then plasma separated viacentrifugation (8000 rpm×6 minutes). The plasma was stored at 20° C.until LC-MS/MS analysis.

Tissue samples: Whisker samples were collected in some animals at 30 hrsafter dosing by hemostats and 10-20 whiskers/animal were placed in a 1.5mL eppendorf tube that contains 200 μL of M-Per buffer (Pierce catalog #78503/78501/78505). The samples were frozen in dry ice/methanol within 1hour of plucking.

It was determined that the mono-amino acid substituted compounds ofFormula I when administered to rats show some conversion tocycloastragenol and the di-amino acid substituted compounds may show aminor amount of conversion to monosubstituted compounds.

Percentage of bioavailability was calculated.

TABLE 4 Study and Bioavailability in Rats Dose Test Level DosingBioavailability compound (mg/kg) Vehicle route % F 2 10 2% EtOH/98%water PO 22.6 4 10 2% EtOH/98% water PO 36 7 10 2% EtOH/98% water PO 4412  10 2% EtOH/98% water PO 27 14  10 5% PEG400/5% solutol PO 93HS-15/90% water 20a, 20b 10 2% EtOH/98% water PO 0.68

Biological Example 5 Administration of Compounds to Male Beagle Dogs andAnalysis of Plasma Levels and Telomerase Activity in Tissues

The plasma level of a compound following a single intravenous and oraladministration in male Beagle dogs was determined. Plasma samples werecollected and used to determine the plasma concentration of the compoundand metabolites. In addition tissue samples, including whisker samplesand PBMC cells were collected for telomerase activity analysis.

Male Beagle dogs were divided into treatment groups according to Table5. The dogs were provided ad libitum SLAC-MO1 #W080701 (ShanghaiLaboratories Animal Center, Shanghai, China) throughout the in-lifeportion of the study with the exception of the overnight fasting periodprior to oral dosing. Water was available ad libitum.

Environmental controls for the animal room were set to maintain atemperature of 23±2° C., humidity of 50-70%, and a 12-hour light/12-hourdark cycle. The 12-hour dark cycle may be temporarily interrupted toaccommodate study procedures. Animals were acclimated to studyprocedures for 1-7 days prior to initial dose administration.

Animals used in this study were selected based on body weights that fallwithin ±20% of the mean body weight, overall health and acclimation tocaging. Animals were given free access to both food and water during thewhole course of study with the exception of the overnight fasting periodprior to oral dosing.

The compounds were dissolved in 2% EtOH/98% water of a solution of 5%PEG400, 5% solutol HS-15 (BASF, TX) 90% water to yield a finalconcentration of 2.5 mg/ml and 1 mg/ml for both intravenous and oraladministration, respectively. The concentration of each compound wasconfirmed by HPLC analysis.

Doses were administered intravenously via the left femoral vein and thenby oral dosing one week later. Body weights were taken on the day ofdose administration. Dose volume was determined based on individual bodyweight taken on day of dosing.

Blood samples (approximately 250 μL) were collected via right femoralvein into tubes containing K2-EDTA anticoagulant and 1 mg/ml NaF atappropriate time points. Blood was stored on ice and then plasmaseparated via centrifugation (8000 rpm×6 minutes). The plasma was storedat 20° C. until LC-MS/MS analysis.

Percentage of bioavailability was calculated and is shown in Table 5.

TABLE 5 Study and Bioavailability in Dogs Dose Test Level DosingBioavailability compound (mg/kg) Vehicle route % F 2 10 2% EtOH/98%water PO 3 4 10 2% EtOH/98% water PO 47 14 10 5% PEG400/5% solutol PO 55HS-15/90% water

Biological Example 6 Upregulation of Bone Marrow HematopoieticStem/Progenitor Cell Telomerase and Cell Proliferation

Human bone marrow-derived CD34+ hematopoietic progenitor cells wereobtained from a 47 year old healthy donor.

i) Telomerase Activation by Compound 4 in Short-Term Liquid Human CellCulture

Human bone marrow-derived CD34+ hematopoietic progenitor cells weregrown in Iscove's Modified Dulbecco's Medium (IMDM) (Invitrogen, CA)+10%fetal bovine serum (FBS) for 3 days in the presence of compound 4 (1 uM,100 nM, 10 nM), vehicle (1% DMSO), or nothing. Telomerase activityincreased by ˜60-70% in the 100 nM compound 4 sample relative to thevehicle control (as assessed by traditional gel-TRAP assay).

TABLE 6 Telomerase activity of huCD34+ cells: (Fold of Vehicle Control)Telomerase activity increase  1 uM compound 4 1.3-fold 100 nM compound 41.8-fold  10 nM compound 4 1.4-fold

ii) Increase in Number of Colony Forming Units in Compound 4-TreatedHuman Cell Cultures (14 Days of Treatment)

Human hematopoietic progenitor CD34+ cells (47 year old healthy donor)were plated into a standard colony formation assay in the presence ofcompound 4 (100 nM), vehicle (0.1% DMSO), or nothing. After 14 days,hematopoietic colonies were enumerated (CFU-E, BFU-3, CFU-GM, andCFU-GEMM). The plates containing compound 4 had 17% more colony formingunits than vehicle alone. (Total colony counts were: untreated, 106.5;vehicle-treated, 103.5; compound 4-treated, 121.5)

TABLE 7 Colony formation of huCD34+ cells: CFU-E BFU-E CFU-GM CFU-GEMMTotal 0.1% DMSO 5.5 16 79.5 2.5 103.5 vehicle 100 nM 11 25.5 80 5 121.5compound 4

Mouse bone marrow-derived lineage-depleted cells (enriched forhematopoietic stem and progenitor cells but not a pure population) wereobtained.

i) Increase in Number of Colony Forming Units in Compound 4-TreatedMouse Bone Marrow Derived Cell Cultures from Normal Wild-Type Mice HSC(112 Days of Treatment)

Wild-type mouse lineage-depleted bone marrow cells from two separatemice were plated into a standard colony formation assay in the presenceof compound 4 (100 nM and 500 nM), vehicle (0.1% DMSO), or nothing.After 12 days, hematopoietic colonies were enumerated (BFU-E, CFU-GM,and CFU-GEMM).

In Mouse 1, total colony counts were: untreated, 136; vehicle-treated,122; 100 nM compound 4, 161; 500 nM compound 4, 162.

In Mouse 2, total colony counts were untreated, 107; vehicle-treated,117; 100 nM compound 4, 121; 500 nM compound 4, 129.

TABLE 8 Colony formation of mouse cells: BFU-E CFU-GM CFU-GEMM TotalMouse 1 0.1% DMSO 41 80 1 122 vehicle Mouse 1 100 nM 50 111 0 161compound 4 Mouse 1 500 nM 38 124 1 162 compound 4 Mouse 2 0.1% DMSO 2689 2 117 vehicle Mouse 2 100 nM 31 90 1 121 compound 4 Mouse 2 500 nM 2998 2 129 compound 4

An increase in total colony counts was observed with administration ofcompound 4.

ii) Telomerase Activity by Compound 4 in Short-Term Liquid Culture inMouse Bone Marrow-Derived Lineage-Depleted Cells from mTERT HeterozygousMice and Wild-Type control (from the same parents)

Lineage-depleted bone marrow cells from mTERT heterozygous and wild typemice were grown in IMDM+15% FBS containing stem cell factor (Kitl),IL-3, and IL-11 for three days in the presence of compound 4 (1 uM, 100nM, or 10 nM), vehicle (0.1% DMSO), or nothing. Telomerase activity inthe wild-type cells increased by 40-50% when treated with 100 nM and 1uM compound 4, relative to the vehicle-treated control.

Telomerase activity in the mTERT heterozygous cells increased by 50%when treated with 1 uM compound 4, relative to the vehicle-treatedcontrol.

iii) Increase in Number of Colony-Forming Units in Control 4-TreatedCultures of mTERT Heterozygous Mouse Cells (12 Days of Treatment).

mTERT heterozygous mouse lineage-depleted bone marrow cells were platedinto a standard colony formation assay in the presence of compound 4(100 nm and 500 nM), vehicle (0.1% DMSO), or nothing. After 12 days,hematopoietic colonies were enumerated (BFU-E, CFU-GM, and CFU-GEMM).Total colony counts were: untreated, 67; vehicle-treated, 64; 100 nMcompound 4, 68; and 500 nM compound 4, 77.

TABLE 9 Compound 4 promotes colony-forming units in Lineage- depletedbone marrow cells from mTERT +/− mice BFU-E CFU-E CFU-GEMM Total 0.1%DMSO 26 37 1 64 100 nM 22 43 3 68 compound 4 500 nM 27 48 1 77 compound4

Biological Example 7 Effect of Compound 4 Administration to BALB/c Miceon Telomerase Activity and Capillary Density in Matrigel Plugs andTelomerase Activity in Bone Marrow Stem/Progenitor Cells

BALB/c mice (2-3 months) were dosed with compound 4 in 2% ethanol at 10mg/kg/day PO (BID). Mice were pre-dosed for 1 day (Day −1). Matrigel™(BDBiosciences, California) was injected subcutaneously in the abdomenon Day 0, and the Matrigel™ plugs were harvested on Day 12.

One half of the plug was analyzed for telomerase activity (RBC cellbuffer extraction, followed by M-PER extraction) using the TRAP assay. A1.9-fold increase (p<0.2) n=5/group in telomerase activity was observedin the Matrigel™ plug after treatment with compound 4.

Total RNA, which reflects cell number, was increased 1.6-fold (p<0.2)n=5/group in Matrigel™ plugs after treatment with compound 4.

The other half of the Matrigel™ plug was used for histology and CD31immunostaining to analyze capillary density (CD31 is a marker forendothelial cells, which line the capillaries). A 1.3-fold increase incapillary density in (CD-31 immuno staining) (p<0.5) n=5/group wasobserved after treatment with compound 4.

ii) Bone Marrow Cells Harvested

Bone marrow stem and progenitor cells were purified from the treatedmice using lineage depletion magnetic sorting technology (Miltenyi MACScolumns). A 1.3 to 1.5-fold increase in telomerase activity asdetermined by the TRAP assay was observed in bone marrow stem andprogenitor cells (p<0.1) n=3/group which had been treated with compound4 as compared to the control.

Biological Example 8 Effect of Compound 4 Administration to C57BL/6Aging TERT (+/−) Mice on Telomerase Activity and Capillary density inMatrigel™ Plugs and Telomerase Activity and Number of Bone MarrowStem/Progenitor Cells

Aging Tert (+/−) mice on a C57BL/6 background (8-9 months) were dosedwith compound 4 at 10 mg/kg/day PO (BID) in 2% ethanol. Mice werepre-dosed for 1 day (Day −1). Matrigel™ was injected subcutaneously inthe abdomen on Day 0, and plugs were harvested on Day 12.

One half of the plug was analyzed for telomerase activity and hemoglobincontent, which is indicative of blood vessel formation (RBC cell bufferextraction, followed by M-PER extraction). The other half of the plugwas processed for histology.

The Matrigel™ plug has a 1.8-fold increase (p<0.02) or a 2.6-foldincrease (p<0.01), in telomerase activity as determined by 2 repeat TRAPexperiments n=15/group for mice treated with compound 4.

The Matrigel™ plug has a 1.2-fold increase in hemoglobin levels (p<0.2)n=15/group for mice treated with compound 4.

Total RNA, which reflects cell number, was increased 1.5-fold (p<0.1)n−15/group in Matrigel™ plugs after treatment with compound 4.

Bone marrow stem and progenitor cells were purified using lineagedepletion magnetic sorting technology (Miltenyi MACS columns). The bonemarrow showed a 1.3-fold increase (p<0.18) or a 1.9-fold increase(p<0.03) in telomerase activity as determined by 2 repeat TRAPexperiments n=6/group in the mice treated with compound 4.

The number of purified bone marrow stem/progenitor cells increased1.5-fold (p<0.1) n=6/group in mice treated with compound 4.

Biological Example 9 Effect of Compound 4 and Compound 7 Administrationon Human Brain Pericytes

Human brain pericytes (27 year old female donor) at PD 10 were culturedfor a total of 30 hr in 0.5 μM compound 7 dissolved in water. Telomeraseactivity and tube formation were analyzed.

Brain pericytes were first cultured for 24 hr in a T-75 flask in 0.5 μMcompound 7, and then split onto a 24 well plate coated with Matrigel inorder to promote tube formation (done in triplicate). 0.5 μM compound 7was again included in the medium. After 6 hr the samples were fixed andbranch points were counted using a microscope, 5 fields/well, with 3wells/condition. Compound 7 treated pericytes had 1.9 times more branchpoints than the control (p<0.15).

Cells were prepared and treated like above, but the 24 well plate wasnot coated with Matrigel. After 6 hr the cells were harvested for TRAPanalysis (M-PER extract). A 2.8-fold increase in telomerase activity wasobserved in compound 7 treated pericytes.

Human brain pericytes (27 year old female donor) at PD 10 were platedand treated with 0.1 and 0.5 μM compound 4 in 0.1% DMSO 24 hr afterseeding. Cells were incubated with drug for 30 hr and harvested for TRAPanalysis (M-PER extract). A 1.8 and 1.9-fold increase in telomeraseactivity was observed with treatment of 0.1 and 0.5 μM, compound 4respectively. Duplicate samples were tested.

Biological Example 10 Effect of Compounds 4 and 12 Administration onHuman Small Airway Epithelial Cells

Human small airway epithelia cells (SAECs) and airway derivedfibroblasts (including the fetal lung fibroblast cell line IMR-90) wereused for in vitro experiments to test the effect of compounds ontelomerase activity.

SAECs and airway derived fibroblast cell line IMR-90 were seeded in 24well plates. They were treated with 1 μM or 0.1 μM of compound 12 for 48hours in a final concentration of 0.2% ethanol in the medium. The cellswere washed with PBS and lysed with M-Per lysis buffer. Gel TRAP assaywas performed to evaluated telomerase activity. It was found thecompound 12 selectively up-regulated telomerase activity 2-4 fold in theepithelium derived cells (SAEC) but not in the fibroblast derived cellIMR-90. Replicate experiments confirmed these findings. In similarstudies Compound 4 had similar properties and potency to compound 12.

SAECs were treated continuously with 0.1 μM of compound 4 in a finalconcentration of 0.004% ethanol for 60 days in continuous culture.Compound 4 increased the long-term replicative capacity of SAECs byabout 2 population doublings (4× increase in calculated cell number). Noeffect was seen in lung fibroblasts in the long-term culture withcompound 4.

Human SAECs or human fibroblasts were grown in the presence of differentconcentrations of compound 4 in a final concentration of 1% DMSO in themedium. After 3 days the cells were harvested and proliferation wasmeasured using the Alamar Blue Proliferation Assay. The SAECs showedincreased proliferation by about 50% in short term culture experiments.No effect was seen with compound 4 treatment in lung fibroblasts onshort-term proliferation. The senescence markers p16 and p21 weresignificantly reduced in SAECs that were treated with compound 4 foronly 3 days, and the reduction of these markers in fibroblasts was verysmall.

SAECs were seeded in a 24 well plate and treated with compound 12 at 1μM and 0.1 μM in a final concentration of 0.2% ethanol in the medium.After 24 hours, media was changed and cells were again treated withcompound 12. In addition a portion of the cells were treated withbleomycin (10 ug/ml) and TGFβ (10 ng/ml). Forty-eight hours after thesecond treatment, cells were washed and lysed with M-Per lysis buffer. Agel TRAP assay was run to evaluate the telomerase activity in the cells.In an in vitro model of fibrosis using TGFβ and bleomycin treated SAECs,myofibroblast/fibrosis biomarker alpha-smooth muscle actin (aSMA)increased and the epithelial biomarker E-cadherin (E-CAD) expressiondecreased. Both TGFβ and bleomycin suppressed SAEC telomerase activityand addition of compound 4 partially restored or protected telomeraseactivity against effects of these compounds in culture.

Although the invention has been described with respect to particularembodiments and applications, those skilled in the art will appreciatethe range of applications and method of the invention disclosed herein.

It is claimed:
 1. A compound of formula I:

wherein X¹, is selected from keto, hydroxy, and

wherein X² is selected from keto, hydroxy, and

wherein X³ is selected from keto, hydroxy, and

wherein at least one of X¹, X², and X³ are

respectively; wherein R¹ or R² are independently selected from—CH(CH₃)₂, and —CH(CH₃)CH₂CH₃ and pharmaceutically acceptable saltsthereof.
 2. The compound of claim 1, wherein X¹ is

wherein R¹ is selected from the group consisting of —CH(CH₃)₂ or—CH(CH₃)CH₂CH₃.
 3. The compound of claim 1, wherein X² is

wherein R² is selected from the group consisting of —CH(CH₃)₂ or—CH(CH₃)CH₂CH₃.
 4. The compound of claim 1, wherein X³ is


5. The compound of claim 1, wherein at X¹, X² or X³ is


6. The compound of claim 1, wherein both X¹ and X² are


7. The compound of claim 1, wherein X¹ or X² is—OC(O)CH(NH₂)CH(CH₃)CH₂CH₃.
 8. The compound of claim 1, wherein both X¹and X² are —OC(O)CH(NH₂)CH(CH₃)CH₂CH₃.
 10. The compound of claim 1,wherein X¹ is a —OC(O)CH(NH₂)CH(CH₃)₂ and X² and X³ are OH.
 11. Thecompound of claim 1, wherein X¹ is —OC(O)CH(NH₂)CH(CH₃)CH₂CH₃ and X² andX³ are —OH.
 12. The compound of claim 1, wherein X² is—OC(O)CH(NH₂)CH(CH₃)₂ and X¹ and X³ are OH.
 13. The compound of claim 1,wherein X² is —OC(O)CH(NH₂)CH(CH₃)CH₂CH₃ and X¹ and X³ are OH.
 14. Acompound selected from the group consisting of:

and pharmaceutically acceptable salts thereof.
 15. The compound of claim14 wherein the pharmaceutically acceptable salt is hydrochloride salt.16. A compound selected from the group consisting of:2-(L)-amino-3-methyl-butyric acid6α,16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester; 2-(L)-amino-3-methyl-butyric acid6α-(2-amino-3-methyl-butyryloxy)-16β-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester; 2-(L)-tert-butoxycarbonylamino-3-methyl-butyric acid3b-acetoxy-16b-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-6a-ylester; 2-(L),3-dimethyl-pentanoic acid6α,16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester, 2-(L)-Amino-3-methyl-butyric acid,16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-3-oxo-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-6α-ylester; 2-(L)-Amino-3-methyl-pentanoic acid6α-(2-amino-3-methyl-pentanoyloxy)-16β-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester; 2-(L)-Amino-3-methyl-butyric acid3β,6α-dihydroxy-16β-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-16β-ylester; and pharmaceutically acceptable salts thereof.
 17. The compoundof claim 16 wherein the pharmaceutically acceptable salt ishydrochloride salt.
 18. The compound of claim 17 wherein the compound isselected from the group consisting of: 2-(L)-amino-3-methyl-butyric acid6α,16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester hydrochloride salt; 2-(L)-amino-3-methyl-butyric acid6α-(2-amino-3-methyl-butyryloxy)-16β-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester hydrochloride salt;2-(L)-tert-butoxycarbonylamino-3-methyl-butyric acid3b-acetoxy-16b-hydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-6a-ylester hydrochloride salt; and 2-(L),3-dimethyl-pentanoic acid6α,16β-dihydroxy-17-[5-(1-hydroxy-1-methyl-ethyl)-2-methyl-tetrahydro-furan-2-yl]-4,4,13,14-tetramethyl-tetradecahydro-cyclopropa[9,10]cyclopenta[a]phenanthren-3β-ylester hydrochloride salt.
 19. A method of increasing telomerase activityin a cell or tissue comprising contacting said cell or tissue with anisolated compound of claim
 1. 20. The method of claim 19 wherein thecell or tissue is identified as requiring increased telomerase activity.21. A pharmaceutical composition comprising the compound of claim 1 in apharmaceutically acceptable vehicle.
 22. The composition of claim 21,wherein said compound is present in said composition at a concentrationof at least 0.1% (w/v).
 23. The pharmaceutical composition comprising atopical formulation of the compound of claim
 1. 24. The composition ofclaim 23, wherein said topical formulation comprises one or morecomponents selected from the group consisting of an emulsifier, athickener, a carrier, and a skin emollient.
 25. A method of enhancingreplicative capacity of cells in vitro or ex vivo, comprising contactingsaid cells with the compound of claim 1, in an amount effect to increasetelomerase activity in said cells.
 26. The method of claim 25, whereinsaid cells are explant cells obtained from a patient.